Monomethylvaline compounds capable of conjugation to ligands

ABSTRACT

Auristatin peptides, including MeVal-Val-Dil-Dap-Norephedrine (MMAE) and MeVal-Val-Dil-Dap-Phe (MMAF), were prepared and attached to Ligands through various linkers, including maleimidocaproyl-val-cit-PAB. The resulting ligand drug conjugates were active in vitro and in vivo.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/098,391 filed Apr. 29, 2011, which is a continuation of U.S. patentapplication Ser. No. 11/833,954 filed Aug. 3, 2007 (now U.S. Pat. No.7,994,135), which is a divisional of U.S. patent application Ser. No.10/983,340 filed Nov. 5, 2004 (now U.S. Pat. No. 7,498,298), which is anapplication claiming the benefit under 35 U.S.C. §119(e) of U.S.Provisional Patent Application No. 60/518,534, filed Nov. 6, 2003, andU.S. Provisional Patent Application No. 60/557,116, filed Mar. 26, 2004,and U.S. Provisional Patent Application No. 60/598,899, filed Aug. 4,2004, and U.S. Provisional Patent Application No. 60/622,455, filed Oct.27, 2004, each of which is incorporated herein by reference in itsentirety.

JOINT RESEARCH AGREEMENT

Some of the subject matter in this application was made by or on behalfof Seattle Genetics, Inc. and Genentech, Inc. as a result of activitiesundertaken within the scope of a joint research agreement effective onor before the date the claimed invention was made.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDERFEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

NOT APPLICABLE

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

The sequence information in the paper copy of the Sequence Listing filedherewith is identical to the sequence information in the only computerreadable form which was filed on Apr. 29, 2011 in application Ser. No.13/098,391 filed Apr. 29, 2011. A request for Transfer of a ComputerReadable Form Under 37 C.F.R. §1.821(e) accompanies this filing.

FIELD OF THE INVENTION

The present invention is directed to a Drug Compound and moreparticularly to Drug-Linker-Ligand Conjugates, Drug-Linker Compounds,and Drug-Ligand Conjugates, to compositions including the same, and tomethods for using the same to treat cancer, an autoimmune disease or aninfectious disease. The present invention is also directed toantibody-drug conjugates, to compositions including the same, and tomethods for using the same to treat cancer, an autoimmune disease or aninfectious. disease. The invention also relates to methods of usingantibody-drug conjugate compounds for in vitro, in situ, and in vivodiagnosis or treatment of mammalian cells, or associated pathologicalconditions.

BACKGROUND OF THE INVENTION

Improving the delivery of drugs and other agents to target cells,tissues and tumors to achieve maximal efficacy and minimal toxicity hasbeen the focus. of considerable research for many years. Though manyattempts have been made to develop effective methods for importingbiologically active molecules into cells, both in vivo and in vitro,none has proved to be entirely satisfactory. Optimizing the associationof the drug with its intracellular target, while minimizingintercellular redistribution of the drug, e.g., to neighboring cells, isoften difficult or inefficient.

Most agents currently administered to a patient parenterally are nottargeted, resulting in systemic delivery of the agent to cells andtissues of the body where it is unnecessary, and often undesirable. Thismay result in adverse drug side effects, and often limits the dose of adrug (e.g., chemotherapeutic (anti-cancer), cytotoxic, enzyme inhibitoragents and antiviral or antimicrobial drugs) that can be administered.By comparison, although oral administration of drugs is considered to bea convenient and economical mode of administration, it shares the sameconcerns of non-specific toxicity to unaffected cells once the drug hasbeen absorbed into the systemic circulation. Further complicationsinvolve problems with oral bioavailability and residence of drug in thegut leading to additional exposure of gut to the drug and hence risk ofgut toxicities. Accordingly, a major goal has been to develop methodsfor specifically targeting agents to cells and tissues. The benefits ofsuch treatment include avoiding the general physiological effects ofinappropriate delivery of such agents to other cells and tissues, suchas uninfected cells. Intracellular targeting may be achieved by methods,compounds and formulations which allow accumulation or retention ofbiologically active agents, i.e. active metabolites, inside cells.

Monoclonal antibody therapy has been established for the targetedtreatment of patients with cancer, immunological and angiogenicdisorders.

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, e.g., drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drg. Del. Rev.26:151-172; U.S. Pat. No. 4,975,278) theoretically allows targeteddelivery of the drug moiety to tumors, and intracellular accumulationtherein, while systemic administration of these unconjugated drug agentsmay result in unacceptable levels of toxicity to normal cells as well asthe tumor cells sought to be eliminated (Baldwin et al., 1986, Lancetpp. (Mar. 15, 1986):603-05; Thorpe, 1985, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies'84: Biological And Clinical Applications, A. Pinchera et al. (ed.s),pp. 475-506). Maximal efficacy with minimal toxicity is sought thereby.Both polyclonal antibodies and monoclonal antibodies have been reportedas useful in these strategies (Rowland et al., 1986, Cancer Immunol.Immunother. 21:183-87). Drugs used in these methods include daunomycin,doxorubicin, methotrexate, and vindesine (Rowland et al., 1986, supra).Toxins used in antibody-toxin conjugates include bacterial toxins suchas diphtheria toxin, plant toxins such as ricin, small molecule toxinssuch as geldanamycin (Kerr et al., 1997, Bioconjugate Chem.8(6):781-784; Mandler et al. (2000) Jour. of the Nat. Cancer Inst.92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791),maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA93:8618-8623), and calicheamicin (Lode et al. (1998) Cancer Res.58:2928; Hinman et al. (1993) Cancer Res. 53:3336-3342). The toxins mayaffect their cytotoxic and cytostatic effects by mechanisms includingtubulin binding, DNA binding, or topoisomerase inhibition (Meyer, D. L.and Senter, P. D. “Recent Advances in Antibody Drug Conjugates forCancer Therapy” in Annual Reports in Medicinal Chemistry, Vol 38 (2003)Chapter 23, 229-237). Some cytotoxic drugs tend to be inactive or lessactive when conjugated to large antibodies or protein receptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al. (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al. (2002) Blood 99(12):4336-42; Witzig et al.(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al. (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The same maytansinoid drug moiety, DM1, was linkedthrough a non-disulfide linker, SMCC, to a mouse murine monoclonalantibody, TA.1 (Chari et al. (1992) Cancer Research 52:127-131). Thisconjugate was reported to be 200-fold less potent than the correspondingdisulfide linker conjugate. The SMCC linker was considered therein to be“noncleavable.”

Several short peptidic compounds have been isolated from the marinemollusc Dolabella auricularia and found to have biological activity(Pettit et al. (1993) Tetrahedron 49:9151; Nakamura et al. (1995)Tetrahedron Letters 36:5059-5062; Sone et al. (1995) Jour. Org Chem.60:4474). Analogs of these compounds have also been prepared, and somewere found to have biological activity (for a review, see Pettit et al.(1998) Anti-Cancer Drug Design 13:243-277). For example, auristatin E(U.S. Pat. No. 5,635,483) is a synthetic analogue of the marine naturalproduct Dolastatin 10, an agent that inhibits tubulin polymerization bybinding to the same domain on tubulin as the anticancer drug vincristine(G. R. Pettit, (1997) Prog. Chem. Org. Nat. Prod. 70:1-79). Dolastatin10, auristatin PE, and auristatin E are linear peptides having fouramino acids, three of which are unique to the dolastatin class ofcompounds, and a C-terminal amide.

The auristatin peptides, auristain E (AE) and monomethylauristatin(MMAE), synthetic analogs of dolastatin, were conjugated to: (i)chimeric monoclonal antibodies cBR96 (specific to Lewis Y oncarcinomas); (ii) cAC10 which is specific to CD30 on hematologicalmalignancies (Klussman, et al. (2004), Bioconjugate Chemistry15(4):765-773; Doronina et al. (2003) Nature Biotechnology21(7):778-784; “Monomethylvaline Compounds Capable of Conjugation toLigands”; Francisco et al. (2003) Blood 102(4):1458-1465; U.S.Publication 2004/0018194; (iii) anti-CD20 antibodies such as RITUXAN®(WO 04/032828) for the treatment of CD20-expressing cancers and immunedisorders; (iv) anti-EphB2 antibodies 2H9 and anti-IL-8 for treatment ofcolorectal cancer (Mao, et al. (2004) Cancer Research 64(3):781-788);(v) E-selectin antibody (Bhaskar et al. (2003) Cancer Res.63:6387-6394); and (vi) other anti-CD30 antibodies (WO 03/043583).

Auristatin E conjugated to monoclonal antibodies are disclosed in Senteret al, Proceedings of the American Association for Cancer Research,Volume 45, Abstract Number 623, presented Mar. 28, 2004.

Despite in vitro data for compounds of the dolastatin class and itsanalogs, significant general toxicities at doses required for achievinga therapeutic effect compromise their efficacy in clinical studies.Accordingly, there is a clear need in the art for dolastatin/auristatinderivatives having significantly lower toxicity, yet useful therapeuticefficiency. These and other limitations and problems of the past areaddressed by the present invention. The ErbB family of receptor tyrosinekinases are important mediators of cell growth, differentiation andsurvival. The receptor family includes four distinct members includingepidermal growth factor receptor (EGFR, ErbB1, HER1), HER2 (ErbB2 orp185^(neu)), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). A panel ofanti-ErbB2 antibodies has been characterized using the human breasttumor cell line SKBR3 (Hudziak et al., (1989) Mol. Cell. Biol.9(3):1165-1172. Maximum inhibition was obtained with the antibody called4D5 which inhibited cellular proliferation by 56%. Other antibodies inthe panel reduced cellular proliferation to a lesser extent in thisassay. The antibody 4D5 was further found to sensitizeErbB2-overexpressing breast tumor cell lines to the cytotoxic effects ofTNF-α (U.S. Pat. No. 5,677,171). The anti-ErbB2 antibodies discussed inHudziak et al. are further characterized in Fendly et al. (1990) CancerResearch 50:1550-1558; Kotts et al. (1990) In vitro 26(3):59A; Sarup etal. (1991) Growth Regulation 1:72-82; Shepard et al. J. (1991) Clin.Immunol. 11(3):117-127; Kumar et al. (1991) Mol. Cell. Biol.11(2):979-986; Lewis et al. (1993) Cancer Immunol. Immunother.37:255-263; Pietras et al. (1994) Oncogene 9:1829-1838; Vitetta et al.(1994) Cancer Research 54:5301-5309; Sliwkowski et al. (1994) J. Biol.Chem. 269(20):14661-14665; Scott et al. (1991) J. Biol. Chem.266:14300-5; D'souza et al. Proc. Natl. Acad. Sci. (1994) 91:7202-7206;Lewis et al. (1996) Cancer Research 56:1457-1465; and Schaefer et al.(1997) Oncogene 15:1385-1394.

Other anti-ErbB2 antibodies with various properties have been describedin Tagliabue et al. Int. J. Cancer 47:933-937 (1991); McKenzie et al.Oncogene 4:543-548 (1989); Maier et al. Cancer Res. 51:5361-5369 (1991);Bacus et al. Molecular Carcinogenesis 3:350-362 (1990); Stancovski etal. Proc. Natl. Acad. Sci. USA 88:8691-8695 (1991); Bacus et al. CancerResearch 52:2580-2589 (1992); Xu et al. Int. J. Cancer 53:401-408(1993); WO94/00136; Kasprzyk et al. Cancer Research 52:2771-2776 (1992);Hancock et al. (1991) Cancer Res. 51:4575-4580; Shawver et al. (1994)Cancer Res. 54:1367-1373; Arteaga et al. (1994) Cancer Res.54:3758-3765; Harwerth et al. (1992) J. Biol. Chem. 267:15160-15167;U.S. Pat. No. 5,783,186; and Klapper et al. (1997) Oncogene14:2099-2109.

Homology screening has resulted in the identification of two other ErbBreceptor family members; ErbB3 (U.S. Pat. No. 5,183,884; U.S. Pat. No.5,480,968; Kra U. S. et al. (1989) Proc. Natl. Acad. Sci. USA86:9193-9197) and ErbB4 (EP 599274; Plowman et al. (1993) Proc. Natl.Acad. Sci. USA 90:1746-1750; and Plowman et al. (1993) Nature366:473-475). Both of these receptors display increased expression on atleast some breast cancer cell lines.

HERCEPTIN® (Trastuzumab) is a recombinant DNA-derived humanizedmonoclonal antibody that selectively binds with high affinity in acell-based assay (Kd=5 nM) to the extracellular domain of the humanepidermal growth factor receptor2 protein, HER2 (ErbB2) (U.S. Pat. No.5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213; U.S. Pat.No. 6,639,055; Coussens L, et al. (1985) Science 230:1132-9; Slamon D J,et al. (1989) Science 244:707-12). Trastuzumab is an IgG1 kappa antibodythat contains human framework regions with thecomplementarity-determining regions of a murine antibody (4D5) thatbinds to HER2. Trastuzumab binds to the HER2 antigen and thus. inhibitsthe growth of cancerous cells. Because Trastuzumab is a humanizedantibody, it minimizes any HAMA response in patients. The humanizedantibody against HER2 is produced by a mammalian cell (Chinese HamsterOvary, CHO) suspension culture. The HER2 (or c-erbB2) proto-oncogeneencodes a transmembrane receptor protein of 185 kDa, which isstructurally related to the epidermal growth factor receptor. HER2protein overexpression is observed in 25%-30% of primary breast cancersand can be determined using an immunohistochemistry based assessment offixed tumor blocks (Press M F, et al. (1993) Cancer Res 53:4960-70.Trastuzumab has been shown, in both in vitro assays and in animals, toinhibit the proliferation of human tumor cells that overexpress HER2(Hudziak R M, et al. (1989) Mol Cell Biol 9:1165-72; Lewis G D, et al.(1993) Cancer Immunol Immunother; 37:255-63; Baselga J, et al. (1998)Cancer Res. 58:2825-2831). Trastuzumab is a mediator ofantibody-dependent cellular cytotoxicity, ADCC (Hotaling T E, et al.(1996) [abstract]. Proc. Annual Meeting Am Assoc Cancer Res; 37:471;Pegram M D, et al. (1997) [abstract]. Proc Am Assoc Cancer Res; 38:602).In vitro, Trastuzumab mediated ADCC has been shown to be preferentiallyexerted on HER2 overexpressing cancer cells compared with cancer cellsthat do not overexpress HER2. HERCEPTIN® as a single agent is indicatedfor the treatment of patients with metastatic breast cancer whose tumorsoverexpress the HER2 protein and who have received one or morechemotherapy regimens for their metastatic disease.

HERCEPTIN® in combination with paclitaxel is indicated for treatment ofpatients with metastatic breast cancer whose tumors overexpress the HER2protein and who have not received chemotherapy for their metastaticdisease. HERCEPTIN® is clinically active in patients withErbB2-overexpressing metastatic breast cancers that have receivedextensive prior anti-cancer therapy (Baselga et al, (1996) J. Clin.Oncol. 14:737-744).

The murine monoclonal anti-HER2 antibody inhibits the growth of breastcancer cell lines that overexpress HER2 at the 2+ and 3+ (1−2×10⁶ HER2receptors per cell) level, but has no activity on cells that expresslower levels of HER2 (Lewis et al., (1993) Cancer Immunol. Immunother.37:255-263). Based on this observation, antibody 4D5 was humanized(huMAb4D5-8, rhuMAb HER2, U.S. Pat. No. 5,821,337; Carter et al., (1992)Proc. Natl. Acad. Sci. USA 89: 4285-4289) and tested in breast cancerpatients whose tumors overexpress HER2 but who had progressed afterconventional chemotherapy (Cobleigh et al., (1999) J. Clin. Oncol. 17:2639-2648).

Although HERCEPTIN is a breakthrough in treating patients withErbB2-overexpressing breast cancers that have received extensive prioranti-cancer therapy, some patients in this population fail to respond orrespond only poorly to HERCEPTIN treatment.

Therefore, there is a significant clinical need for developing furtherHER2-directed cancer therapies for those patients withHER2-overexpressing tumors or other diseases associated with HER2expression that do not respond, or respond poorly, to HERCEPTINtreatment. The recitation of any reference in this application is not anadmission that the reference is prior art to this application.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides Drug-Linker-Ligandcompounds having the Formula Ia:

LA_(a)-W_(w)—Y_(y)-D)_(p)  Ia

-   -   or a pharmaceutically acceptable salt or solvate thereof    -   wherein,    -   L- is a Ligand unit;    -   -A_(a)-W_(w)—Y_(y)— is a Linker unit (LU), wherein the Linker        unit includes:    -   -A- is a Stretcher unit,    -   a is 0 or 1,    -   each —W— is independently an Amino Acid unit,    -   w is an integer ranging from 0 to 12,    -   —Y— is a Spacer unit, and    -   y is 0, 1 or 2;    -   p ranges from 1 to about 20; and    -   -D is a Drug unit having the Formulas D_(E) and D_(F):

-   -   wherein, independently at each location:    -   R² is selected from H and C₁-C₈ alkyl;    -   R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁵ is selected from H and methyl;    -   or R⁴ and R⁵ jointly form a carbocyclic ring and have the        formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from H, C₁-C₈ alkyl and C₃-C₈ carbocycle        and n is selected from 2, 3, 4, 5 and 6;    -   R⁶ is selected from H and C₁-C₈ alkyl;    -   R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈        carbocycle and O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and C₁-C₈ alkyl;    -   R¹⁰ is selected from aryl or C₃-C₈ heterocycle;    -   Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is C₂-C₈ alkyl;    -   R¹⁴ is H or C₁-C₈ alkyl;    -   each occurrence of R¹⁵ is independently H, COON,        —(CH₂)_(n)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈        alkyl;    -   each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH; where; n is an integer ranging from 0 to 6; and    -   R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈        heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle).

In another aspect, Drug Compounds having the Formula Ib are provided:

or pharmaceutically acceptable salts or solvates thereof,

-   -   wherein:    -   R² is selected from hydrogen and —C₁-C₈ alkyl;    -   R³ is selected from hydrogen, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈        heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from hydrogen, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle),        —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle) wherein        R⁵ is selected from —H and -methyl; or R⁴ and R⁵ jointly, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the carbon atom to which they are attached;    -   R⁶ is selected from H and —C₁-C₈ alkyl;    -   R⁷ is selected from H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, aryl,        —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈        heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and —C₁-C₈ alkyl;    -   R¹⁹ is selected from aryl group or —C₃-C₈ heterocycle;    -   Z is —O—, —S—, —NH—, or —NR¹²—, wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, —C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is —C₂-C₈ alkyl;    -   R¹⁴ is H or —C₁-C₈ alkyl;    -   each occurrence of R¹⁵ is independently H, —COOH,        —(CH₂)_(n)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈        alkyl;    -   each occurrence of R¹⁶ is independently H, —C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH; and    -   n is an integer ranging from 0 to 6.

The compounds of Formula (Ib) are useful for treating cancer, anautoimmune disease or an infectious disease in a patient or useful as anintermediate for the synthesis of a Drug-Linker, Drug-Linker-LigandConjugate, and Drug-Ligand Conjugate having a cleavable Drug unit.

In another aspect, compositions are provided including an effectiveamount of a Drug-Linker-Ligand Conjugate and a pharmaceuticallyacceptable carrier or vehicle.

In still another aspect, the invention provides pharmaceuticalcompositions comprising an effective amount of a Drug-Linker Compoundand a pharmaceutically acceptable carrier or vehicle.

In still another aspect, the invention provides compositions comprisingan effective amount of a Drug-Ligand Conjugate having a cleavable Drugunit from the Drug-Ligand Conjugate and a pharmaceutically acceptablecarrier or vehicle.

In yet another aspect, the invention provides methods for killing orinhibiting the multiplication of a tumor cell or cancer cell includingadministering to a patient in need thereof an effective amount of aDrug-Linker Compound.

In another aspect, the invention provides methods for killing orinhibiting the multiplication of a tumor cell or cancer cell includingadministering to a patient in need thereof an effective amount of aDrug-Linker-Ligand Conjugate.

In another aspect, the invention provides methods for killing orinhibiting the multiplication of a tumor cell or cancer cell includingadministering to a patient in need thereof an effective amount of aDrug-Ligand Conjugate having a cleavable Drug unit from the Drug-LigandConjugate.

In still another aspect, the invention provides methods for treatingcancer including administering to a patient in need thereof an effectiveamount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for treatingcancer including administering to a patient in need thereof an effectiveamount of a Drug-Linker-Ligand Conjugate.

In yet another aspect, the invention provides methods for treatingcancer including administering to a patient in need thereof an effectiveamount of a Drug-Ligand Conjugate having a cleavable Drug unit from theDrug-Ligand Conjugate.

In still another aspect, the invention provides methods for killing orinhibiting the replication of a cell that expresses an autoimmuneantibody including administering to a patient in need thereof aneffective amount of a Drug-Linker Compound.

In another aspect, the invention provides methods for killing orinhibiting the replication of a cell that expresses an autoimmuneantibody including administering to a patient in need thereof aneffective amount of a Drug-Linker-Ligand Conjugate.

In another aspect, the invention provides methods for killing orinhibiting the replication of a cell that expresses an autoimmuneantibody including administering to a patient in need thereof aneffective amount of a Drug-Ligand Conjugate having a cleavable Drug unitfrom the Drug-Ligand Conjugate.

In yet another aspect, the invention provides methods for treating anautoimmune disease including administering to a patient in need thereofan effective amount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for treating anautoimmune disease including administering to a patient in need thereofan effective amount of a Drug-Linker-Ligand Conjugate.

In yet another aspect, the invention provides methods for treating anautoimmune disease including administering to a patient in need thereofan effective amount of a Drug-Ligand Conjugate having a cleavable Drugunit from the Drug-Ligand Conjugate.

In still another aspect, the invention provides methods for treating aninfectious. Disease including administering to a patient in need thereofan effective amount of a Drug-Linker Compound.

In still another aspect, the invention provides methods for treating aninfectious disease including administering to a patient in need thereofan effective amount of a Drug-Linker-Ligand Conjugate.

In still another aspect, the invention provides methods for treating aninfectious disease including administering to a patient in need thereofan effective amount of a Drug-Ligand Conjugate having a cleavable Drugunit from the Drug-Ligand Conjugate.

In yet another aspect, the invention provides methods for preventing themultiplication of a tumor cell or cancer cell including administering toa patient in need thereof an effective amount of a Drug-Linker Compound.

In another aspect, the invention provides methods for preventing themultiplication of a tumor cell or cancer cell including administering toa patient in need thereof an effective amount of a Drug-Linker-LigandConjugate.

In another aspect, the invention provides methods for preventing themultiplication of a tumor cell or cancer cell including administering toa patient in need thereof an effective amount of a Drug-Ligand Conjugatehaving a cleavable Drug unit from the Drug-Ligand Conjugate.

In still another aspect, the invention provides methods for preventingcancer including administering to a patient in need thereof an effectiveamount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for preventingcancer including administering to a patient in need thereof an effectiveamount of a Drug-Linker-Ligand Conjugate.

In yet another aspect, the invention provides methods for preventingcancer including administering to a patient in need thereof an effectiveamount of a Drug-Ligand Conjugate having a cleavable Drug unit from theDrug-Ligand Conjugate.

In still another aspect, the invention provides methods for preventingthe multiplication of a cell that expresses an autoimmune antibodyincluding administering to a patient in need thereof an effective amountof a Drug-Linker Compound.

In another aspect, the invention provides methods for preventing themultiplication of a cell that expresses an autoimmune antibody includingadministering to a patient in need thereof an effective amount of aDrug-Linker-Ligand Conjugate.

In another aspect, the invention provides methods for preventing themultiplication of a cell that expresses an autoimmune antibody includingadministering to a patient in need thereof an effective amount of aDrug-Ligand Conjugate having a cleavable Drug unit from the Drug-LigandConjugate.

In yet another aspect, the invention provides methods for preventing anautoimmune disease including administering to a patient in need thereofan effective amount of a Drug-Linker Compound.

In yet another aspect, the invention provides methods for preventing anautoimmune disease including administering to a patient in need thereofan effective amount of a Drug-Linker-Ligand Conjugate.

In yet another aspect, the invention provides methods for preventing anautoimmune disease including administering to a patient in need thereofan effective amount of a Drug-Ligand Conjugate having a cleavable Drugunit from the Drug-Ligand Conjugate.

In still another aspect, the invention provides methods for preventingan infectious disease including administering to a patient in needthereof an effective amount of a Drug-Linker Compound.

In still another aspect, the invention provides methods for preventingan infectious disease including administering to a patient in needthereof an effective amount of a Drug-Linker-Ligand Conjugate.

In still another aspect, the invention provides methods for preventingan infectious disease including administering to a patient in needthereof an effective amount of a Drug-Ligand Conjugate having acleavable Drug unit from the Drug-Ligand Conjugate.

In another aspect, a Drug Compound is provided which can be used as anintermediate for the synthesis of a Drug-Linker Compound having acleavable Drug unit from the Drug-Ligand Conjugate.

In another aspect, a Drug-Linker Compound is provided which can be usedas an intermediate for the synthesis of a Drug-Linker-Ligand Conjugate.

In another aspect, compounds having Formula Ia′ are provided:

AbA _(a)-W_(w)—Y_(y)-D)_(p)  Ia′

-   -   or a pharmaceutically acceptable salt or solvate thereof,        wherein:        -   Ab includes an antibody including one which binds to CD30,            CD40, CD70, and Lewis Y antigen,    -   A is a Stretcher unit,    -   a is 0 or 1,    -   each W is independently an Amino Acid unit,    -   w is an integer ranging from 0 to 12,    -   Y is a Spacer unit, and    -   y is 0, 1 or 2,    -   p ranges from 1 to about 20, and    -   D is a Drug unit selected from Formulas D_(E) and D_(F):

-   -   wherein, independently at each location:    -   R² is selected from H and C₁-C₈ alkyl;    -   R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁵ is selected from H and methyl;    -   or R⁴ and R⁵ jointly form a carbocyclic ring and have the        formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from H, C₁-C₈ alkyl and C₃-C₈ carbocycle        and n is selected from 2, 3, 4, 5 and 6;    -   R⁶ is selected from H and C₁-C₈ alkyl;    -   R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈        carbocycle and O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and C₁-C₈ alkyl;    -   R¹⁰ is selected from aryl or C₃-C₈ heterocycle;    -   Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is C₂-C₈ alkyl;    -   R¹⁴ is H or C₁-C₈ alkyl;    -   each occurrence of R¹⁵ is independently H, COOH,        —(CH₂)_(n)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈        alkyl;    -   each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH;    -   R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈        heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and    -   n is an integer ranging from 0 to 6.

In one embodiment, Ab is not an antibody which binds to an ErbB receptoror which binds to one or more of receptors (1)-(35):

-   -   (1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank        accession no. NM_(—)001203);    -   (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_(—)003486);    -   (3) STEAP1 (six transmembrane epithelial antigen of prostate,        Genbank accession no. NM_(—)012449);    -   (4) 0772P (CA125, MUC16, Genbank accession no. AF361486);    -   (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,        mesothelin, Genbank accession no. NM_(—)005823);    -   (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34        (sodium phosphate), member 2, type II sodium-dependent phosphate        transporter 3b, Genbank accession no. NM_(—)006424);    -   (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,        Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats        (type 1 and type 1-like), transmembrane domain I and short        cytoplasmic domain, (emaphoring) 5B, Genbank accession no.        AB040878);    -   (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN Cdna        2700050C12, RIKEN Cdna 2700050C12 gene, Genbank accession no.        AY358628);    -   (9) ETBR (Endothelin type B receptor, Genbank accession no.        AY275463);    -   (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank        accession no. NM_(—)017763);    -   (11) STEAP2 (HGNC_(—)8639, IPCA-1, PCANAP1, STAMP1, STEAP2,        STMP, prostate cancer associated gene 1, prostate cancer        associated protein 1, six transmembrane epithelial antigen of        prostate 2, six transmembrane prostate protein, Genbank        accession no. AF455138);    -   (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor        potential cation channel, subfamily M, member 4, Genbank        accession no. NM_(—)017636);    -   (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,        teratocarcinoma-derived growth factor, Genbank accession no.        NP_(—)003203 or NM_(—)003212);    -   (14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr        virus receptor) or Hs.73792, Genbank accession no. M26004);    -   (15) CD79b (Igb (immunoglobulin-associated beta), B29, Genbank        accession no. NM_(—)000626);    -   (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing        phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank        accession no. NM_(—)030764);    -   (17) HER2 (Genbank accession no. M11730);    -   (18) NCA (Genbank accession no. M18728);    -   (19) MDP (Genbank accession no. BC017023);    -   (20) IL20Rα (Genbank accession no. AF184971);    -   (21) Brevican (Genbank accession no. AF229053);    -   (22) Ephb2R (Genbank accession no. NM_(—)004442);    -   (23) ASLG659 (Genbank accession no. AX092328);    -   (24) PSCA (Genbank accession no. AJ297436);    -   (25) GEDA (Genbank accession no. AY260763);    -   (26) BAFF-R (Genbank accession no. NP_(—)443177.1);    -   (27) CD22 (Genbank accession no. NP-001762.1);    -   (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a B        cell-specific protein that covalently interacts with Ig beta        (CD79B) and forms a complex on the surface with Ig M molecules,        transduces a signal involved in B-cell differentiation, Genbank        accession No. NP_(—)001774.1);    -   (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled        receptor that is activated by the CXCL13 chemokine, functions in        lymphocyte migration and humoral defense, plays a role in HIV-2        infection and perhaps development of AIDS, lymphoma, myeloma,        and leukemia, Genbank accession No. NP_(—)001707.1);    -   (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)        that binds peptides and presents them to CD4+ T lymphocytes,        Genbank accession No. NP_(—)002111.1);    -   (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5,        an ion channel gated by extracellular ATP, may be involved in        synaptic transmission and neurogenesis, deficiency may        contribute to the pathophysiology of idiopathic detrusor        instability, Genbank accession No. NP_(—)002552.2);    -   (32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank        accession No. NP_(—)001773.1);    -   (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane        protein of the leucine rich repeat (LRR) family, regulates        B-cell activation and apoptosis, loss of function is associated        with increased disease activity in patients with systemic lupus        erythematosis, Genbank accession No. NP_(—)005573.1);    -   (34) FCRH1 (Fc receptor-like protein 1, a putative receptor for        the immunoglobulin Fc domain that contains C2 type Ig-like and        ITAM domains, may have a role in B-lymphocyte differentiation,        Genbank accession No. NP_(—)443170.1); or    -   (35) IRTA2 (Immunoglobulin superfamily receptor translocation        associated 2, a putative immunoreceptor with possible roles in B        cell development and lymphomagenesis; deregulation of the gene        by translocation occurs in some B cell malignancies, Genbank        accession No. NP_(—)112571.1).

In still another aspect, the invention provides pharmaceuticalcompositions comprising an effective amount of a Drug-Linker-AntibodyConjugate and a pharmaceutically acceptable carrier or vehicle.

In still another aspect, the invention provides compositions comprisingan effective amount of a Drug-Antibody Conjugate having a cleavable Drugunit (moiety) from the Drug-Antibody Conjugate and a pharmaceuticallyacceptable carrier or vehicle.

In another aspect, the invention provides methods for killing orinhibiting the multiplication of a tumor cell or cancer cell includingadministering to a patient in need thereof an effective amount of aDrug-Linker-Antibody Conjugate.

In another aspect, the invention provides methods for killing orinhibiting the multiplication of a tumor cell or cancer cell includingadministering to a patient in need thereof an effective amount of aDrug-Antibody Conjugate having a cleavable Drug unit from theDrug-Antibody Conjugate.

In yet another aspect, the invention provides methods for treatingcancer including administering to a patient in need thereof an effectiveamount of a Drug-Linker-Antibody Conjugate.

In yet another aspect, the invention provides methods for treatingcancer including administering to a patient in need thereof an effectiveamount of a Drug-Antibody Conjugate having a cleavable Drug unit fromthe Drug-Antibody Conjugate.

In another aspect, the invention provides methods for killing orinhibiting the replication of a cell that expresses an autoimmuneantibody including administering to a patient in need thereof aneffective amount of a Drug-Linker-Antibody Conjugate.

In another aspect, the invention provides methods for killing orinhibiting the replication of a cell that expresses an autoimmuneantibody including administering to a patient in need thereof aneffective amount of a Drug-Antibody Conjugate having a cleavable Drugunit from the Drug-Antibody Conjugate.

In yet another aspect, the invention provides methods for treating anautoimmune disease including administering to a patient in need thereofan effective amount of a Drug-Linker-Antibody Conjugate.

In yet another aspect, the invention provides methods for treating anautoimmune disease including administering to a patient in need thereofan effective amount of a Drug-Antibody Conjugate having a cleavable Drugunit from the Drug-Antibody Conjugate.

In still another aspect, the invention provides methods for treating aninfectious disease including administering to a patient in need thereofan effective amount of a Drug-Linker-Antibody Conjugate.

In still another aspect, the invention provides methods for treating aninfectious disease including administering to a patient in need thereofan effective amount of a Drug-Antibody Conjugate having a cleavable Drugunit from the Drug-Antibody Conjugate.

In another aspect, the invention provides methods for preventing themultiplication of a tumor cell or cancer cell including administering toa patient in need thereof an effective amount of a Drug-Linker-AntibodyConjugate.

In another aspect, the invention provides methods for preventing themultiplication of a tumor cell or cancer cell including administering toa patient in need thereof an effective amount of a Drug-AntibodyConjugate having a cleavable Drug unit from the Drug-Antibody Conjugate.

In yet another aspect, the invention provides methods for preventingcancer including administering to a patient in need thereof an effectiveamount of a Drug-Linker-Antibody Conjugate.

In yet another aspect, the invention provides methods for preventingcancer including administering to a patient in need thereof an effectiveamount of a Drug-Antibody Conjugate having a cleavable Drug unit fromthe Drug-Antibody Conjugate.

In another aspect, the invention provides methods for preventing themultiplication of a cell that expresses an autoimmune antibody includingadministering to a patient in need thereof an effective amount of aDrug-Linker-Antibody Conjugate.

In another aspect, the invention provides methods for preventing themultiplication of a cell that expresses an autoimmune antibody includingadministering to a patient in need thereof an effective amount of aDrug-Antibody Conjugate having a cleavable Drug unit from theDrug-Antibody Conjugate.

In yet another aspect, the invention provides methods for preventing anautoimmune disease including administering to a patient in need thereofan effective amount of a Drug-Linker-Antibody Conjugate.

In yet another aspect, the invention provides methods for preventing anautoimmune disease including administering to a patient in need thereofan effective amount of a Drug-Antibody Conjugate having a cleavable Drugunit from the Drug-Antibody Conjugate.

In still another aspect, the invention provides methods for preventingan infectious disease including administering to a patient in needthereof an effective amount of a Drug-Linker-Antibody Conjugate.

In still another aspect, the invention provides methods for preventingan infectious disease including administering to a patient in needthereof an effective amount of a Drug-Antibody Conjugate having acleavable Drug unit from the Drug-Antibody Conjugate.

In another aspect, a Drug Compound is provided which can be used as anintermediate for the synthesis of a Drug-Linker Compound having acleavable Drug unit from the Drug-Antibody Conjugate.

In another aspect, a Drug-Linker Compound is provided which can be usedas an intermediate for the synthesis of a Drug-Linker-AntibodyConjugate.

In one aspect, the present invention provides Drug-Linker-AntibodyConjugates (also referred to as antibody-drug conjugates) having FormulaIc:

AbA _(a)-W_(w)—Y_(y)-D)_(p)  Ic

-   -   or a pharmaceutically acceptable salt or solvate thereof,        wherein:    -   Ab is an antibody which binds to one or more of the antigens        (1)-(35):    -   (1) BMPR1B (bone morphogenetic protein receptor-type OB, Genbank        accession no. NM_(—)001203);    -   (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_(—)003486);    -   (3) STEAP1 (six transmembrane epithelial antigen of prostate,        Genbank accession no. NM_(—)012449);    -   (4) 0772P (CA125, MUC16, Genbank accession no. AF361486);    -   (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,        mesothelin, Genbank accession no. NM_(—)005823);    -   (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34        (sodium phosphate), member 2, type II sodium-dependent phosphate        transporter 3b, Genbank accession no. NM_(—)006424);    -   (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,        Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats        (type 1 and type 1-like), transmembrane domain I and short        cytoplasmic domain, (emaphoring) 5B, Genbank accession no.        AB040878);    -   (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN Cdna        2700050C12, RIKEN Cdna 2700050C12 gene, Genbank accession no.        AY358628);    -   (9) ETBR (Endothelin type B receptor, Genbank accession no.        AY275463);    -   (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank        accession no. NM_(—)017763);    -   (11) STEAP2 (HGNC_(—)8639, IPCA-1, PCANAP1, STAMP1, STEAP2,        STMP, prostate cancer associated gene 1, prostate cancer        associated protein 1, six transmembrane epithelial antigen of        prostate 2, six transmembrane prostate protein, Genbank        accession no. AF455138);    -   (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor        potential cation channel, subfamily M, member 4, Genbank        accession no. NM_(—)017636);    -   (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,        teratocarcinoma-derived growth factor, Genbank accession no.        NP_(—)003203 or NM_(—)003212);    -   (14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr        virus receptor) or Hs.73792, Genbank accession no. M26004);    -   (15) CD79b (10 (immunoglobulin-associated beta), B29, Genbank        accession no. NM_(—)000626);    -   (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing        phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank        accession no. NM_(—)030764);    -   (17) HER2 (Genbank accession no. M11730);    -   (18) NCA (Genbank accession no. M18728);    -   (19) MDP (Genbank accession no. BC017023);    -   (20) IL20Rα (Genbank accession no. AF184971);    -   (21) Brevican (Genbank accession no. AF229053);    -   (22) Ephb2R (Genbank accession no. NM_(—)004442);    -   (23) ASLG659 (Genbank accession no. AX092328);    -   (24) PSCA (Genbank accession no. AJ297436);    -   (25) GEDA (Genbank accession no. AY260763);    -   (26) BAFF-R (Genbank accession no. NP_(—)443177.1);    -   (27) CD22 (Genbank accession no. NP-001762.1);    -   (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a B        cell-specific protein that covalently interacts with Ig beta        (CD79B) and forms a complex on the surface with Ig M molecules,        transduces a signal involved in B-cell differentiation, Genbank        accession No. NP_(—)001774.1);    -   (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled        receptor that is activated by the CXCL13 chemokine, functions in        lymphocyte migration and humoral defense, plays a role in HIV-2        infection and perhaps development of AIDS, lymphoma, myeloma,        and leukemia, Genbank accession No. NP_(—)001707.1);    -   (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)        that binds peptides and presents them to CD4+ T lymphocytes,        Genbank accession No. NP_(—)002111.1);    -   (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5,        an ion channel gated by extracellular ATP, may be involved in        synaptic transmission and neurogenesis, deficiency may        contribute to the pathophysiology of idiopathic detrusor        instability, Genbank accession No. NP_(—)002552.2);    -   (32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank        accession No. NP_(—)001773.1);    -   (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane        protein of the leucine rich repeat (LRR) family, regulates        B-cell activation and apoptosis, loss of function is associated        with increased disease activity in patients with systemic lupus        erythematosis, Genbank accession No. NP_(—)005573.1);    -   (34) FCRH1 (Fc receptor-like protein 1, a putative receptor for        the immunoglobulin Fc domain that contains C2 type Ig-like and        ITAM domains, may have a role in B-lymphocyte differentiation,        Genbank accession No. NP_(—)443170.1); or    -   (35) IRTA2 (Immunoglobulin superfamily receptor translocation        associated 2, a putative immunoreceptor with possible roles in B        cell development and lymphomagenesis; deregulation of the gene        by translocation occurs in some B cell malignancies, Genbank        accession No. NP_(—)112571.1);    -   A is a Stretcher unit,    -   a is 0 or 1,    -   each W is independently an Amino Acid unit,    -   w is an integer ranging from 0 to 12,    -   Y is a Spacer unit, and    -   y is 0, 1 or 2,    -   p ranges from 1 to about 20, and    -   D is a Drug moiety selected from Formulas D_(E) and D_(F):

-   -   wherein the wavy line of D_(E) and D_(F) indicates the covalent        attachment site to A, W, or Y, and independently at each        location:    -   R² is selected from H and C₁-C₈ alkyl;    -   R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁵ is selected from H and methyl;    -   or R⁴ and R⁵ jointly form a carbocyclic ring and have the        formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from H, C₁-C₈ alkyl and C₃-C₈ carbocycle        and n is selected from 2, 3, 4, 5 and 6;    -   R⁶ is selected from H and C₁-C₈ alkyl;    -   R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈        carbocycle and O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and C₁-C₈ alkyl;    -   R₁₀ is selected from aryl or C₃-C₈ heterocycle;    -   Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,        —(R¹³O)m-R¹⁴, or —(R¹³O)m-CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is C₂-C₈ alkyl;    -   R¹⁴ is H or C₁-C₈ alkyl;    -   each occurrence of R¹⁵ is independently H, COOH,        —(CH₂)n-N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;    -   each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or        —(CH₂), —COOH;    -   R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈        heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and    -   n is an integer ranging from 0 to 6.

In another aspect, the antibody of the antibody-drug conjugate (ADC) ofthe invention specifically binds to a receptor encoded by an ErbB2 gene.

In another aspect, the antibody of the antibody-drug conjugate is ahumanized antibody selected from huMAb4D5-1, huMAb4D5-2, huMAb4D5-3,huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8(Trastuzumab).

In another aspect, the invention includes an article of manufacturecomprising an antibody-drug conjugate compound of the invention; acontainer; and a package insert or label indicating that the compoundcan be used to treat cancer characterized by the overexpression of anErbB2 receptor.

In another aspect, the invention includes a method for the treatment ofcancer in a mammal, wherein the cancer is characterized by theoverexpression of an ErbB2 receptor and does not respond, or respondspoorly, to treatment with an anti-ErbB2 antibody, comprisingadministering to the mammal a therapeutically effective amount of anantibody-drug conjugate compound of the invention.

In another aspect, a substantial amount of the drug moiety is notcleaved from the antibody until the antibody-drug conjugate compoundenters a cell with a cell-surface receptor specific for the antibody ofthe antibody-drug conjugate, and the drug moiety is cleaved from theantibody when the antibody-drug conjugate does enter the cell.

In another aspect, the bioavailability of the antibody-drug conjugatecompound or an intracellular metabolite of the compound in a mammal isimproved when compared to a drug compound comprising the drug moiety ofthe antibody-drug conjugate compound, or when compared to an analog ofthe compound not having the drug moiety.

In another aspect, the drug moiety is intracellularly cleaved in amammal from the antibody of the compound, or an intracellular metaboliteof the compound.

In another aspect, the invention includes a pharmaceutical compositioncomprising an effective amount of the antibody-drug conjugate compoundof the invention, or a pharmaceutically acceptable salt thereof, and apharmaceutically acceptable diluent, carrier or excipient. Thecomposition may further comprise a therapeutically effective amount ofchemotherapeutic agent such as a tubulin-forming inhibitor, atopoisomerase inhibitor, and a DNA binder.

In another aspect, the invention includes a method for killing orinhibiting the proliferation of tumor cells or cancer cells comprisingtreating tumor cells or cancer cells with an amount of the antibody-drugconjugate compound of the invention, or a pharmaceutically acceptablesalt or solvate thereof, being effective to kill or inhibit theproliferation of the tumor cells or cancer cells.

In another aspect, the invention includes a method of inhibitingcellular proliferation comprising exposing mammalian cells in a cellculture medium to an antibody drug conjugate compound of the invention,wherein the antibody drug conjugate compound enters the cells and thedrug is cleaved from the remainder of the antibody drug conjugatecompound; whereby proliferation of the cells is inhibited.

In another aspect, the invention includes a method of treating cancercomprising administering to a patient a formulation of an antibody-drugconjugate compound of the invention and a pharmaceutically acceptablediluent, carrier or excipient.

In another aspect, the invention includes an assay for detecting cancercells comprising:

-   -   (a) exposing cells to an antibody-drug conjugate compound of the        invention; and    -   (b) determining the extent of binding of the antibody-drug        conjugate compound to the cells.

The invention will best be understood by reference to the followingdetailed description of the exemplary embodiments, taken in conjunctionwith the accompanying drawings, figures, and schemes. The discussionbelow is descriptive, illustrative and exemplary and is not to be takenas limiting the scope defined by any appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an in vivo, single dose, efficacy assay of cAC10-mcMMAF insubcutaneous Karpas-299 ALCL xenografts.

FIG. 2 shows an in vivo, single dose, efficacy assay of cAC10-mcMMAF insubcutaneous L540cy. For this study there were 4 mice in the untreatedgroup and 10 in each of the treatment groups.

FIGS. 3 a and 3 b show in vivo efficacy of cBR96-mcMMAF in subcutaneousL2987. The filed triangles in FIG. 3 a and arrows in FIG. 3 b indicatethe days of therapy.

FIGS. 4 a and 4 b show in vitro activity ofcAC10-antibody-drugconjugates against CD30⁺ cell lines.

FIGS. 5 a and 5 b show in vitro activity of cBR96-antibody-drugconjugates against Le^(y+) cell lines.

FIGS. 6 a and 6 b show in vitro activity of c1F6-antibody-drugconjugates against CD70⁺ renal cell carcinoma cell lines.

FIG. 7 shows an in vitro, cell proliferation assay with SK-BR-3 cellstreated with antibody drug conjugates (ADC): --Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, -∘- Trastuzumab-MC-MMAF, 4.1MMAF/Ab, and -Δ- Trastuzumab-MC-MMAF, 4.8 MMAF/Ab, measured in RelativeFluorescence Units (RLU) versus μg/ml concentration of ADC.H=Trastuzumab where H is linked via a cysteine [cys].

FIG. 8 shows an in vitro, cell proliferation assay with BT-474 cellstreated with ADC: -- Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, -∘-Trastuzumab-MC-MMAF, 4.1 MMAF/Ab, and -Δ- Trastuzumab-MC-MMAF, 4.8MMAF/Ab.

FIG. 9 shows an in vitro, cell proliferation assay with MCF-7 cellstreated with ADC: -- Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab, -∘-Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab, and -Δ-Trastuzumab-MC-MMAF, 4.1 MMAF/Ab.

FIG. 10 shows an in vitro, cell proliferation assay with MDA-MB-468cells treated with ADC: -- Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab, -∘-Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab, and -Δ-Trastuzumab-MC-vc-PAB-MMAF, 3.7 MMAF/Ab.

FIG. 11 shows a plasma concentration clearance study afteradministration of H-MC-vc-PAB-MMAF-TEG and H-MC-vc-PAB-MMAF toSprague-Dawley rats: The administered dose was 2 mg of ADC per kg ofrat. Concentrations of total antibody and ADC were measured over time.(H=Trastuzumab).

FIG. 12 shows a plasma concentration clearance study afteradministration of H-MC-vc-MMAE to Cynomolgus monkeys at different doses:0.5, 1.5, 2.5, and 3.0 mg/kg administered at day 1 and day 21.Concentrations of total antibody and ADC were measured over time.(H=Trastuzumab).

FIG. 13 shows the mean tumor volume change over time in athymic nudemice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with:Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 μg/m²) andTrastuzumab-MC-vc-PAB-MMAF (555 μg/m²). (H=Trastuzumab).

FIG. 14 shows the mean tumor volume change over time in athymic nudemice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 with 10mg/kg (660 μg/m²) of Trastuzumab-MC-MMAE and 1250 μg/m²Trastuzumab-MC-vc-PAB-MMAE.

FIG. 15 shows the mean tumor volume change over time in athymic nudemice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 withVehicle and 650 μg/m² trastuzumab-MC-MMAF.

FIG. 16 shows the mean tumor volume change over time in athymic nudemice with MMTV-HER2 Fo5 Mammary tumor allografts dosed on Day 0 withVehicle and 350 μg/m² of four trastuzumab-MC-MMAF conjugates where theMMAF/trastuzumab (H) ratio is 2, 4, 5.9 and 6.

FIG. 17 shows the Group mean change, with error bars, in animal (rat)body weights (Mean±SD) after administration of Vehicle,trastuzumab-MC-val-cit-MMAF, trastuzumab-MC(Me)-val-cit-PAB-MMAF,trastuzumab-MC-MMAF and trastuzumab-MC-val-cit-PAB-MMAF.

FIG. 18 shows the Group mean change in animal (rat) body weights(Mean±SD) after administration of 9.94 mg/kg H-MC-vc-MMAF, 24.90 mg/kgH-MC-vc-MMAF, 10.69 mg/kg H-MC(Me)-vc-PAB-MMAF, 26.78 mg/kgH-MC(Me)-vc-PAB-MMAF, 10.17 mg/kg H-MC-MMAF, 25.50 mg/kg H-MC-MMAF, and21.85 mg/kg H-MC-vc-PAB-MMAF. H=trastuzumab. The MC linker is attachedvia a cysteine of trastuzumab for each conjugate.

FIG. 19 shows the Group mean change, with error bars, in Sprague Dawleyrat body weights (Mean±SD) after administration of trastuzumab(H)-MC-MMAF at doses of 2105, 3158, and 4210 μg/m². The MC linker isattached via a cysteine of trastuzumab for each conjugate.

FIG. 20 shows examples of compounds with a non self-immolative Spacerunit.

FIG. 21 shows a scheme of a possible mechanism of Drug release from aPAB group which is attached directly to -D via a carbamate or carbonategroup.

FIG. 22 shows a scheme of a possible mechanism of Drug release from aPAB group which is attached directly to -D via an ether or aminelinkage.

FIG. 23 shows an example of a branched spacer unit,bis(hydroxymethyl)styrene (BHMS) unit, which can be used to incorporateand release multiple drug.

FIG. 24 shows a scheme of the CellTiter-Glo® Assay.

FIG. 25 shows the synthesis of an N-terminal tripeptide unit F which isa useful intermediate for the synthesis of the drug compounds of FormulaIb.

FIG. 26 shows the synthesis of an N-terminal tripeptide unit F which isa useful intermediate for the synthesis of the drug compounds of FormulaIb.

FIG. 27 shows the synthesis of an N-terminal tripeptide unit F which isa useful intermediate for the synthesis of the drug compounds of FormulaIb.

FIG. 28 shows the synthesis of useful linkers.

FIG. 29 shows the synthesis of useful linkers.

FIG. 30 shows a general synthesis of an illustrative Linker unitcontaining a maleimide Stretcher group and optionally a p-aminobenzylether self-immolative Spacer.

FIG. 31 shows the synthesis of a val-cit dipeptide Linker having amaleimide Stretcher and optionally a p-aminobenzyl self-immolativeSpacer.

FIG. 32 shows the synthesis of a phe-lys(Mtr) dipeptide Linker unithaving a maleimide Stretcher unit and a p-aminobenzyl self-immolativeSpacer unit.

FIG. 33 shows the synthesis of a Drug-Linker Compound that contains anamide or carbamate group, linking the Drug unit to the Linker unit.

FIG. 34 shows illustrative methods useful for linking a Drug to a Ligandto form a Drug-Linker Compound.

FIG. 35 shows the synthesis of a val-cit dipeptide linker having amaleimide Stretcher unit and a bis(4-hydroxymethyl)styrene (BHMS) unit.

FIG. 36 shows methodology useful for making Drug-Linker-Ligandconjugates having about 2 to about 4 drugs per antibody.

FIG. 37 shows the synthesis of MC-MMAF via t-butyl ester.

FIG. 38 shows the synthesis of MC-MMAF (11) via dimethoxybenzyl ester.

FIG. 39 shows the synthesis of analog of mc-MMAF.

DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

Definitions and Abbreviations

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

When trade names are used herein, applicants intend to independentlyinclude the trade name product formulation, the generic drug, and theactive pharmaceutical ingredient(s) of the trade name product.

The term “antibody” herein is used in the broadest sense andspecifically covers intact monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments, so long as theyexhibit the desired biological activity. An antibody is a proteingenerated by the immune system that is capable of recognizing andbinding to a specific antigen. Described in terms of its structure, anantibody typically has a Y-shaped protein consisting of four amino acidchains, two heavy and two light. Each antibody has primarily tworegions: a variable region and a constant region. The variable region,located on the ends of the arms of the Y, binds to and interacts withthe target antigen. This variable region includes a complementarydetermining region (CDR) that recognizes and binds to a specific bindingsite on a particular antigen. The constant region, located on the tailof the Y, is recognized by and interacts with the immune system(Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology,5th Ed., Garland Publishing, New York). A target antigen generally hasnumerous binding sites, also called epitopes, recognized by CDRs onmultiple antibodies. Each antibody that specifically binds to adifferent epitope has a different structure. Thus, one antigen may havemore than one corresponding antibody.

The term “antibody” as used herein, also refers to a full-lengthimmunoglobulin molecule or an immunologically active portion of afull-length immunoglobulin molecule, i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. In one aspect, however, the immunoglobulin is of human, murine,or rabbit origin. In another aspect, the antibodies are polyclonal,monoclonal, bispecific, human, humanized or chimeric antibodies, singlechain antibodies, Fv, Fab fragments, F(ab′) fragments, F(ab′)₂fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, CDR's, and epitope-bindingfragments of any of the above which immunospecifically bind to cancercell antigens, viral antigens or microbial antigens.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al. (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al. (1991) Nature, 352:624-628 andMarks et al. (1991) J. Mol. Biol., 222:581-597, for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855).

Various methods have been employed to produce monoclonal antibodies(MAbs). Hybridoma technology, which refers to a cloned cell line thatproduces a single type of antibody, uses the cells of various species,including mice (murine), hamsters, rats, and humans. Another method toprepare MAbs uses genetic engineering including recombinant DNAtechniques. Monoclonal antibodies made from these techniques include,among others, chimeric antibodies and humanized antibodies. A chimericantibody combines DNA encoding regions from more than one type ofspecies. For example, a chimeric antibody may derive the variable regionfrom a mouse and the constant region from a human. A humanized antibodycomes predominantly from a human, even though it contains nonhumanportions. Like a chimeric antibody, a humanized antibody may contain acompletely human constant region. But unlike a chimeric antibody, thevariable region may be partially derived from a human. The nonhuman,synthetic portions of a humanized antibody often come from CDRs inmurine antibodies. In any event, these regions are crucial to allow theantibody to recognize and bind to a specific antigen.

As noted, murine antibodies can be used. While useful for diagnosticsand short-term therapies, murine antibodies cannot be administered topeople long-term without increasing the risk of a deleteriousimmunogenic response. This response, called Human Anti-Mouse Antibody(HAMA), occurs when a human immune system recognizes the murine antibodyas foreign and attacks it. A HAMA response can cause toxic shock or evendeath. Chimeric and humanized antibodies reduce the likelihood of a HAMAresponse by minimizing the nonhuman portions of administered antibodies.Furthermore, chimeric and humanized antibodies have the additionalbenefit of activating secondary human immune responses, such as antibodydependent cellular cytotoxicity.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fvfragments; diabodies; linear antibodies; single-chain antibodymolecules; and multispecific antibodies formed from antibodyfragment(s).

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (CL) and heavy chainconstant domains, CH1, CH2 and CH3. The constant domains may be nativesequence constant domains (e.g., human native sequence constant domains)or amino acid sequence variant thereof.

The intact antibody may have one or more “effector functions” whichrefer to those biological activities attributable to the Fc region (anative sequence Fc region or amino acid sequence variant Fc region) ofan antibody. Examples of antibody effector functions include C1qbinding; complement dependent cytotoxicity; Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

The expressions “ErbB2” and “HER2” are used interchangeably herein andrefer to human HER2 protein described, for example, in Semba et al.,Proc. Natl. Acad. Sci. USA, 82:6497-6501 (1985) and Yamamoto et al.,(1986) Nature, 319:230-234 (Genebank accession number X03363). The term“erbB2” refers to the gene encoding human ErbB2 and “neu” refers to thegene encoding rat p185neu. Preferred ErbB2 is native sequence humanErbB2. Antibodies to ErbB receptors are available commercially from anumber of sources, including, for example, Santa Cruz Biotechnology,Inc., California, USA.

By “ErbB ligand” is meant a polypeptide which binds to and/or activatesan ErbB receptor. The ErbB ligand may be a native sequence human ErbBligand such as epidermal growth factor (EGF) (Savage et al. (1972) J.Biol. Chem., 247:7612-7621); transforming growth factor alpha (TGF-α)(Marquardt et al. (1984) Science 223:1079-1082); amphiregulin also knownas schwanoma or keratinocyte autocrine growth factor (Shoyab et al.(1989) Science 243:1074-1076; Kimura et al., Nature, 348:257-260 (1990);and Cook et al., Mol. Cell. Biol., 11:2547-2557 (1991)); betacellulin(Shing et al., Science, 259:1604-1607 (1993); and Sasada et al.,Biochem. Biophys. Res. Commun., 190:1173 (1993)); heparin-bindingepidermal growth factor (HB-EGF) (Higashiyama et al., Science,251:936-939 (1991)); epiregulin (Toyoda et al., J. Biol. Chem.,270:7495-7500 (1995); and Komurasaki et al., Oncogene, 15:2841-2848(1997)); a heregulin (see below); neuregulin-2 (NRG-2) (Carraway et al.,Nature, 387:512-516 (1997)); neuregulin-3 (NRG-3) (Zhang et al., Proc.Natl. Acad. Sci., 94:9562-9567 (1997)); neuregulin-4 (NRG-4) (Harari etal., Oncogene, 18:2681-89 (1999)) or cripto (CR-1) (Kannan et al., J.Biol. Chem., 272(6):3330-3335 (1997)). ErbB ligands which bind EGFRinclude EGF, TGF-α, amphiregulin, betacellulin, HB-EGF and epiregulin.ErbB ligands which bind ErbB3 include heregulins. ErbB ligands capableof binding ErbB4 include betacellulin, epiregulin, HB-EGF, NRG-2, NRG-3,NRG-4 and heregulins. The ErbB ligand may also be a synthetic ErbBligand. The synthetic ligand may be specific for a particular ErbBreceptor, or may recognize particular ErbB receptor complexes. Anexample of a synthetic ligand is the synthetic heregulin/EGF chimerabiregulin (see, for example, Jones et al., (1999) FEBS Letters,447:227-231, which is incorporated by reference). “Heregulin” (HRG)refers to a polypeptide encoded by the heregulin gene product asdisclosed in U.S. Pat. No. 5,641,869 or Marchionni et al., Nature,362:312-318 (1993). Examples of heregulins include heregulin-α,heregulin-β1, heregulin-β2 and heregulin-β3 (Holmes et al., Science,256:1205-1210 (1992); and U.S. Pat. No. 5,641,869); neu differentiationfactor (NDF) (Peles et al., Cell 69: 205-216 (1992)); acetylcholinereceptor-inducing activity (ARIA) (Falls et al. (1993) Cell 72:801-815);glial growth factors (GGFs) (Marchionni et al., Nature, 362:312-318(1993)); sensory and motor neuron derived factor (SMDF) (Ho et al., J.Biol. Chem., 270:14523-14532 (1995)); γ-heregulin (Schaefer et al.,Oncogene, 15:1385-1394 (1997)). The term includes biologically activefragments and/or amino acid sequence variants of a native sequence HRGpolypeptide, such as an EGF-like domain fragment thereof (e.g.,HRGβ1177-244).

“ErbB hetero-oligomer” is a noncovalently associated oligomer comprisingat least two different ErbB receptors. An “ErbB dimer” is anoncovalently associated oligomer that comprises two different ErbBreceptors. Such complexes may form when a cell expressing two or moreErbB receptors is exposed to an ErbB ligand. ErbB oligomers, such asErbB dimers, can be isolated by immunoprecipitation and analyzed bySDS-PAGE as described in Sliwkowski et al., J. Biol. Chem.,269(20):14661-14665 (1994), for example. Examples of such ErbBhetero-oligomers include EGFR-ErbB2 (also referred to as HER1/HER2),ErbB2-ErbB3 (HER2/HER3) and ErbB3-ErbB4 (HER3/HER4) complexes. Moreover,the ErbB hetero-oligomer may comprise two or more ErbB2 receptorscombined with a different ErbB receptor, such as ErbB3, ErbB4 or EGFR(ErbB1). Other proteins, such as a cytokine receptor subunit (e.g.,gp130) may be included in the hetero-oligomer.

A “native sequence” polypeptide is one which has the same amino acidsequence as a polypeptide, e.g., tumor-associated antigen receptor,derived from nature. Such native sequence polypeptides can be isolatedfrom nature or can be produced by recombinant or synthetic means. Thus,a native sequence polypeptide can have the amino acid sequence ofnaturally-occurring human polypeptide, murine polypeptide, orpolypeptide from any other mammalian species.

The term “amino acid sequence variant” refers to polypeptides havingamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants will possess atleast about 70% homology with at least one receptor binding domain of anative ligand, or with at least one ligand binding domain of a nativereceptor, such as a tumor-associated antigen, and preferably, they willbe at least about 80%, more preferably, at least about 90% homologouswith such receptor or ligand binding domains. The amino acid sequencevariants possess substitutions, deletions, and/or insertions at certainpositions within the amino acid sequence of the native amino acidsequence. “Sequence identity” is defined as the percentage of residuesin the amino acid sequence variant that are identical after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Methods and computer programs for thealignment are well known in the art. One such computer program is “Align2,” authored by Genentech, Inc., which was filed with user documentationin the United States Copyright Office, Washington, D.C. 20559, on Dec.10, 1991.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, (1991) Annu. Rev. Immunol, 9:457-92.To assess ADCC activity of a molecule of interest, an in vitro ADCCassay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat.No. 5,821,337 may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA,95:652-656 (1998). The terms “Fc receptor” or “FcR” are used to describea receptor that binds to the Fc region of an antibody. The preferred FcRis a native sequence human FcR. Moreover, a preferred FcR is one whichbinds an IgG antibody (a gamma receptor) and includes receptors of theFcγRI, FcγRII, and FcγRIII subclasses, including allelic variants andalternatively spliced forms of these receptors. FcγRII receptors includeFcγRIIA (an “activating receptor”) and FcγRIIB (an “inhibitingreceptor”), which have similar amino acid sequences that differprimarily in the cytoplasmic domains thereof. Activating receptorFcγRIIA contains an immunoreceptor tyrosine-based activation motif(ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB containsan immunoreceptor tyrosine-based inhibition motif (ITIM) in itscytoplasmic domain. (See review M. in Daëron, Annu. Rev. Immunol.,15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994);and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs,including those to be identified in the future, are encompassed by theterm “FcR” herein. The term also includes the neonatal receptor, FcRn,which is responsible for the transfer of maternal IgGs to the fetus.(Guyer et al., J. Immunol., 117:587 (1976) and Kim et al., J. Immunol.,24:249 (1994)).

“Complement dependent cytotoxicity” or “CDC” refers to the ability of amolecule to lyse a target in the presence of complement. The complementactivation pathway is initiated by the binding of the first component ofthe complement system (Clq) to a molecule (e.g., an antibody) complexedwith a cognate antigen. To assess complement activation, a CDC assay,e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods,202:163 (1996), may be performed.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al. (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.). The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variabledomain; Kabat et al. supra) and/or those residues from a “hypervariableloop” (e.g., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the lightchain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy chain variable domain; Chothia and Lesk (1987) J. Mol. Biol.,196:901-917). “Framework Region” or “FR” residues are those variabledomain residues other than the hypervariable region residues as hereindefined.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-binding sites and is still capable of cross-linkingantigen. “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the VH-VL dimer. Collectively,the six hypervariable regions confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three hypervariable regions specific for an antigen) hasthe ability to recognize and bind antigen, although at a lower affinitythan the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH₁) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(VH) connected to a variable light domain (VL) in the same polypeptidechain (VH-VL). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites. Diabodies are described more fully in, forexample, EP 404,097; WO 93/11161; and Hollinger et al. (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fe), typically that of a humanimmunoglobulin. For further details, see Jones et al. (1986) Nature,321:522-525; Riechmann et al. (1988) Nature 332:323-329; and Presta,(1992) Curr. Op. Struct. Biol., 2:593-596.

Humanized anti-ErbB2 antibodies include huMAb4D5-1, huMAb4D5-2,huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 andhuMAb4D5-8 (HERCEPTIN®) as described in Table 3 of U.S. Pat. No.5,821,337 expressly incorporated herein by reference; humanized 520C9(WO 93/21319) and humanized 2C4 antibodies as described herein below.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody “which binds” an antigen of interest is one capable ofbinding that antigen with sufficient affinity such that the antibody isuseful in targeting a cell expressing the antigen. An antibody which“induces apoptosis” is one which induces programmed cell death asdetermined by binding of annexin V, fragmentation of DNA, cellshrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/orformation of membrane vesicles (called apoptotic bodies). The cell is atumor cell, e.g., a breast, ovarian, stomach, endometrial, salivarygland, lung, kidney, colon, thyroid, pancreatic or bladder cell. Variousmethods are available for evaluating the cellular events associated withapoptosis. For example, phosphatidyl serine (PS) translocation can bemeasured by annexin binding; DNA fragmentation can be evaluated throughDNA laddering; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.

A “disorder” is any condition that would benefit from treatment of thepresent invention. This includes chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question. Non-limiting examples of disorders to betreated herein include benign and malignant tumors; leukemia andlymphoid malignancies, in particular breast, ovarian, stomach,endometrial, salivary gland, lung, kidney, colon, thyroid, pancreatic,prostate or bladder cancer; neuronal, glial, astrocytal, hypothalamicand other glandular, macrophagal, epithelial, stromal and blastocoelicdisorders; and inflammatory, angiogenic and immunologic disorders.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can, for example, be measured by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR). The term“substantial amount” refers to a majority, i.e. >50% of a population, ofa collection or a sample.

The term “intracellular metabolite” refers to a compound resulting froma metabolic process or reaction inside a cell on an antibody drugconjugate (ADC). The metabolic process or reaction may be an enzymaticprocess such as proteolytic cleavage of a peptide linker of the ADC, orhydrolysis of a functional group such as a hydrazone, ester, or amide.Intracellular metabolites include, but are not limited to, antibodiesand free drug which have undergone intracellular cleavage after entry,diffusion, uptake or transport into a cell.

The terms “intracellularly cleaved” and “intracellular cleavage” referto a metabolic process or reaction inside a cell on an Drug-LigandConjugate, a Drug-Linker-Ligand Conjugate, an antibody drug conjugate(ADC) or the like whereby the covalent attachment, e.g., the linker,between the drug moiety (D) and the antibody (Ab) is broken, resultingin the free drug dissociated from the antibody inside the cell. Thecleaved moieties of the Drug-Ligand Conjugate, a Drug-Linker-LigandConjugate or ADC are thus intracellular metabolites. The term“bioavailability” refers to the systemic availability (i.e.,blood/plasma levels) of a given amount of drug administered to apatient. Bioavailability is an absolute term that indicates measurementof both the time (rate) and total amount (extent) of drug that reachesthe general circulation from an administered dosage form.

The term “cytotoxic activity” refers to a cell-killing, cytostatic oranti-proliferation effect of an antibody drug conjugate compound or anintracellular metabolite of an antibody drug conjugate compound.Cytotoxic activity may be expressed as the IC₅₀ value which is theconcentration (molar or mass) per unit volume at which half the cellssurvive.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer.

An “ErbB2-expressing cancer” is one which produces sufficient levels ofErbB2 at the surface of cells thereof, such that an anti-ErbB2 antibodycan bind thereto and have a therapeutic effect with respect to thecancer.

A cancer “characterized by excessive activation” of an ErbB2 receptor isone in which the extent of ErbB2 receptor activation in cancer cellssignificantly exceeds the level of activation of that receptor innon-cancerous cells of the same tissue type. Such excessive activationmay result from overexpression of the ErbB2 receptor and/or greater thannormal levels of an ErbB2 ligand available for activating the ErbB2receptor in the cancer cells. Such excessive activation may cause and/orbe caused by the malignant state of a cancer cell. In some embodiments,the cancer will be subjected to a diagnostic or prognostic assay todetermine whether amplification and/or overexpression of an ErbB2receptor is occurring which results in such excessive activation of theErbB2 receptor. Alternatively, or additionally, the cancer may besubjected to a diagnostic or prognostic assay to determine whetheramplification and/or overexpression an ErbB2 ligand is occurring in thecancer which attributes to excessive activation of the receptor. In asubset of such cancers, excessive activation of the receptor may resultfrom an autocrine stimulatory pathway.

A cancer which “overexpresses” an ErbB2 receptor is one which hassignificantly higher levels of an ErbB2 receptor at the cell surfacethereof, compared to a noncancerous cell of the same tissue type. Suchoverexpression may be caused by gene amplification or by increasedtranscription or translation. ErbB2 receptor overexpression may bedetermined in a diagnostic or prognostic assay by evaluating increasedlevels of the ErbB2 protein present on the surface of a cell (e.g., viaan immunohistochemistry assay; IHC). Alternatively, or additionally, onemay measure levels of ErbB2-encoding nucleic acid in the cell, e.g., viafluorescent in situ hybridization (FISH; see WO 98/45479), southernblotting, or polymerase chain reaction (PCR) techniques, such as realtime quantitative PCR (RT-PCR).

Overexpression of the ErbB2 ligand, may be determined diagnostically byevaluating levels of the ligand (or nucleic acid encoding it) in thepatient, e.g., in a tumor biopsy or by various diagnostic assays such asthe IHC, FISH, southern blotting, PCR or in vivo assays described above.One may also study ErbB2 receptor overexpression by measuring shedantigen (e.g., ErbB2 extracellular domain) in a biological fluid such asserum (see, e.g., U.S. Pat. No. 4,933,294; WO 91/05264; U.S. Pat. No.5,401,638; and Sias et al., (1990) J. Immunol. Methods, 132: 73-80).Aside from the above assays, various other in vivo assays are availableto the skilled practitioner. For example, one may expose cells withinthe body of the patient to an antibody which is optionally labeled witha detectable label, e.g., a radioactive isotope, and binding of theantibody to cells in the patient can be evaluated, e.g., by externalscanning for radioactivity or by analyzing a biopsy taken from a patientpreviously exposed to the antibody.

The tumors overexpressing HER2 are rated by immunohistochemical scorescorresponding to the number of copies of HER2 molecules expressed percell, and can been determined biochemically: 0=0-10,000 copies/cell,1+=at least about 200,000 copies/cell, 2+=at least about 500,000copies/cell, 3+=about 1−2×10⁶ copies/cell. Overexpression of HER2 at the3+ level, which leads to ligand-independent activation of the tyrosinekinase (Hudziak et al., (1987) Proc. Natl. Acad. Sci. USA,84:7159-7163), occurs in approximately 30% of breast cancers, and inthese patients, relapse-free survival and overall survival arediminished (Slamon et al., (1989) Science, 244:707-712; Slamon et al.,(1987) Science, 235:177-182). Conversely, a cancer which is “notcharacterized by overexpression of the ErbB2 receptor” is one which, ina diagnostic assay, does not express higher than normal levels of ErbB2receptor compared to a noncancerous cell of the same tissue type.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e. ²¹¹At,¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰C, and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including synthetic analogs and derivativesthereof. In one aspect, the term is not intended to include radioactiveisotopes.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; TLK 286 (TELCYTA™); acetogenins (especiallybullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acamptothecin (including the synthetic analogue topotecan (HYCAMTIN®),CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;bisphosphonates, such as clodronate; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin gamma1I andcalicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)) and anthracyclines such as annamycin, AD 32,alcarubicin, daunorubicin, dexrazoxane, DX-52-1, epirubicin, GPX-100,idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, anesperamicin, neocarzinostatin chromophore and related chromoproteinenediyne antiobiotic chromophores, aclacinomysins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, carabicin,carminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin,6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin),esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin; folic acid analogues such asdenopterin, pteropterin, and trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide,mitotane, and trilostane; folic acid replenisher such as folinic acid(leucovorin); aceglatone; anti-folate anti-neoplastic agents such asALIMTA®, LY231514 pemetrexed, dihydrofolate reductase inhibitors such asmethotrexate, anti-metabolites such as 5-fluorouracil (5-FU) and itsprodrugs such as UFT, S-1 and capecitabine, and thymidylate synthaseinhibitors and glycinamide ribonucleotide formyltransferase inhibitorssuch as raltitrexed (TOMUDEX®, TDX); inhibitors of dihydropyrimidinedehydrogenase such as eniluracil; aldophosphamide glycoside;aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate;an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan;lonidainine; maytansinoids such as maytansine and ansamitocins;mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin;phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.);razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine(ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol;mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”);cyclophosphamide; thiotepa; taxoids and taxanes, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine)(GEMZAR®); 6-thioguanine; mercaptopurine; platinum; platinumanalogs or platinum-based analogs such as cisplatin, oxaliplatin andcarboplatin; vinblastine)(VELBAN®); etoposide (VP-16); ifosfamide;mitoxantrone; vincristine (ONCOVIN®); vinca alkaloid; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; xeloda;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; pharmaceutically acceptablesalts, acids or derivatives of any of the above; as well as combinationsof two or more of the above such as CHOP, an abbreviation for a combinedtherapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone,and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin(ELOXATIN™) combined with 5-FU and leucovorin.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON® toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those thatinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, andepidermal growth factor receptor (EGF-R); vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

As used herein, the term “EGFR-targeted drug” refers to a therapeuticagent that binds to EGFR and, optionally, inhibits EGFR activation.Examples of such agents include antibodies and small molecules that bindto EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCCCRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et al.)and variants thereof, such as chimerized 225 (C225 or Cetuximab;ERBITUX®) and reshaped human 225 (H225) (see, WO 96/40210, ImcloneSystems Inc.); antibodies that bind type II mutant EGFR (U.S. Pat. No.5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF (see WO 98/50433, Abgenix). The anti-EGFR antibodymay be conjugated with a cyotoxic agent, thus generating animmunoconjugate (see, e.g., EP 659,439A2, Merck Patent GmbH). Examplesof small molecules that bind to EGFR include ZD1839 or Gefitinib(IRESSA™; Astra Zeneca), Erlotinib HCl (CP-358774, TARCEVA™;Genentech/OSI) and AG1478, AG1571 (SU 5271; Sugen).

A “tyrosine kinase inhibitor” is a molecule which inhibits to someextent tyrosine kinase activity of a tyrosine kinase such as an ErbBreceptor. Examples of such inhibitors include the EGFR-targeted drugsnoted in the preceding paragraph as well as quinazolines such as PD153035,4-(3-chloroanilino) quinazoline, pyridopyrimidines,pyrimidopyrimidines, pyrrolopyrimidines, such as CGP 59326, CGP 60261and CGP 62706, and pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines, curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide), tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lambert); antisensemolecules (e.g., those that bind to ErbB-encoding nucleic acid);quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG);pan-ErbB inhibitors such as CI-1033 (Pfizer); Affinitac (ISIS 3521;Isis/Lilly); Imatinib mesylate (Gleevac; Novartis); PKI 166 (Novartis);GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib(Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11(Imclone); or as described in any of the following patent publications:U.S. Pat. No. 5,804,396; WO 99/09016 (American Cyanamid); WO 98/43960(American Cyanamid); WO 97/38983 (Warner Lambert); WO 99/06378 (WarnerLambert); WO 99/06396 (Warner Lambert); WO 96/30347 (Pfizer, Inc); WO96/33978 (Zeneca); WO 96/3397 (Zeneca); and WO 96/33980 (Zeneca).

An “anti-angiogenic agent” refers to a compound which blocks, orinterferes with to some degree, the development of blood vessels. Theanti-angiogenic factor may, for instance, be a small molecule orantibody that binds to a growth factor or growth factor receptorinvolved in promoting angiogenesis. In one embodiment, theanti-angiogenic factor is an antibody that binds to Vascular EndothelialGrowth Factor (VEGF).

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically or hydrolytically activated or converted into themore active parent form. See, e.g., Wilman, “Prodrugs in CancerChemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615thMeeting Belfast (1986) and Stella et al., “Prodrugs: A Chemical Approachto Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al.,(ed.), pp. 247-267, Humana Press (1985). The prodrugs of this inventioninclude, but are not limited to, phosphate-containing prodrugs,thiophosphate-containing prodrugs, sulfate-containing prodrugs,peptide-containing prodrugs, D-amino acid-modified prodrugs,glycosylated prodrugs, β-lactam-containing prodrugs, optionallysubstituted phenoxyacetamide-containing prodrugs or optionallysubstituted phenylacetamide-containing prodrugs, 5-fluorocytosine andother 5-fluorouridine prodrugs which can be converted into the moreactive cytotoxic free drug. Examples of cytotoxic drugs that can bederivatized into a prodrug form for use in this invention include, butare not limited to, those chemotherapeutic agents described above.

A “liposome” is a small vesicle composed of various types of lipids,phospholipids and/or surfactant which is useful for delivery of a drug(such as including the anti-CD30, CD40, CD70 or Lewis Y antibodies and,optionally, a chemotherapeutic agent) to a mammal. The components of theliposome are commonly arranged in a bilayer formation, similar to thelipid arrangement of biological membranes.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe antibody nucleic acid. An isolated nucleic acid molecule is otherthan in the form or setting in which it is found in nature. Isolatednucleic acid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking can be accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers can be used inaccordance with conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

An “autoimmune disease” herein is a disease or disorder arising from anddirected against an individual's own tissues or a co-segregate ormanifestation thereof or resulting condition therefrom. Examples ofautoimmune diseases or disorders include, but are not limited toarthritis (rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis, and ankylosing spondylitis),psoriasis, dermatitis including atopic dermatitis; chronic idiopathicurticaria, including chronic autoimmune urticaria,polymyositis/dermatomyositis, toxic epidermal necrolysis, systemicscleroderma and sclerosis, responses associated with inflammatory boweldisease (IBD) (Crohn's disease, ulcerative colitis), and IBD withco-segregate of pyoderma gangrenosum, erythema nodosum, primarysclerosing cholangitis, and/or episcleritis), respiratory distresssyndrome, including adult respiratory distress syndrome (ARDS),meningitis, IgE-mediated diseases such as anaphylaxis and allergicrhinitis, encephalitis such as Rasmussen's encephalitis, uveitis,colitis such as microscopic colitis and collagenous colitis,glomerulonephritis (GN) such as membranous GN, idiopathic membranous GN,membranous proliferative GN (MPGN), including Type I and Type II, andrapidly progressive GN, allergic conditions, eczema, asthma, conditionsinvolving infiltration of T cells and chronic inflammatory responses,atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency,systemic lupus erythematosus (SLE) such as cutaneous SLE, lupus(including nephritis, cerebritis, pediatric, non-renal, discoid,alopecia), juvenile onset diabetes, multiple sclerosis (MS) such asspino-optical MS, allergic encephalomyelitis, immune responsesassociated with acute and delayed hypersensitivity mediated by cytokinesand T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis includingWegener's granulomatosis, agranulocytosis, vasculitis (including LargeVessel vasculitis (including Polymyalgia Rheumatica and Giant Cell(Takayasu's) Arteritis), Medium Vessel vasculitis (including Kawasaki'sDisease and Polyarteritis Nodosa), CNS vasculitis, and ANCA-associatedvasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), aplasticanemia, Coombs positive anemia, Diamond Blackfan anemia, immunehemolytic anemia including autoimmune hemolytic anemia (AIHA),pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency,hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseasesinvolving leukocyte diapedesis, CNS inflammatory disorders, multipleorgan injury syndrome, myasthenia gravis, antigen-antibody complexmediated diseases, anti-glomerular basement membrane disease,anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease,Castleman's syndrome, Goodpasture's Syndrome, Lambert-Eaton MyasthenicSyndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnsonsyndrome, solid organ transplant rejection (including pretreatment forhigh panel reactive antibody titers, IgA deposit in tissues, andrejection arising from renal transplantation, liver transplantation,intestinal transplantation, cardiac transplantation, etc.), graft versushost disease (GVHD), pemphigoid bullous, pemphigus (including vulgaris,foliaceus, and pemphigus mucus-membrane pemphigoid), autoimmunepolyendocrinopathies, Reiter's disease, stiff-man syndrome, immunecomplex nephritis, IgM polyneuropathies or IgM mediated neuropathy,idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenicpurpura (TTP), thrombocytopenia (as developed by myocardial infarctionpatients, for example), including autoimmune thrombocytopenia,autoimmune disease of the testis and ovary including autoimmune orchitisand oophoritis, primary hypothyroidism; autoimmune endocrine diseasesincluding autoimmune thyroiditis, chronic thyroiditis (Hashimoto'sThyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison'sdisease, Grave's disease, autoimmune polyglandular syndromes (orpolyglandular endocrinopathy syndromes), Type I diabetes also referredto as insulin-dependent diabetes mellitus (IDDM), including pediatricIDDM, and Sheehan's syndrome; autoimmune hepatitis, Lymphoidinterstitial pneumonitis (HIV), bronchiolitis obliterans(non-transplant) vs NSIP, Guillain-Barrè Syndrome, Berger's Disease (IgAnephropathy), primary biliary cirrhosis, celiac sprue (glutenenteropathy), refractory sprue with co-segregate dermatitisherpetiformis, cryoglobulinemia, amylotrophic lateral sclerosis (ALS;Lou Gehrig's disease), coronary artery disease, autoimmune inner eardisease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome(OMS), polychondritis such as refractory polychondritis, pulmonaryalveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis,monoclonal gammopathy of uncertain/unknown significance (MGUS),peripheral neuropathy, paraneoplastic syndrome, channelopathies such asepilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness,periodic paralysis, and channelopathies of the CNS; autism, inflammatorymyopathy, and focal segmental glomerulosclerosis (FSGS).

“Alkyl” is C₁-C₁₈ hydrocarbon containing normal, secondary, tertiary orcyclic carbon atoms. Examples are methyl (Me, —CH₃), ethyl (Et,—CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (i-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃.

“Alkenyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp² double bond. Examples include, but are not limitedto: ethylene or vinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), cyclopentenyl(—C₅H₇), and 5-hexenyl (—CH₂CH₂CH₂CH₂CH═CH₂).

“Alkynyl” is C₂-C₁₈ hydrocarbon containing normal, secondary, tertiaryor cyclic carbon atoms with at least one site of unsaturation, i.e. acarbon-carbon, sp triple bond. Examples include, but are not limited to:acetylenic (—C≡CH) and propargyl (—CH₂C≡CH).

“Alkylene” refers to a saturated, branched or straight chain or cyclichydrocarbon radical of 1-18 carbon atoms, and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkyleneradicals include, but are not limited to: methylene (—CH₂—) 1,2-ethyl(—CH₂CH₂—), 1,3-propyl (—CH₂CH₂CH₂—), 1,4-butyl (—CH₂CH₂CH₂CH₂—), andthe like.

“Alkenylene” refers to an unsaturated, branched or straight chain orcyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkene. Typicalalkenylene radicals include, but are not limited to: 1,2-ethylene(—CH═CH—). “Alkynylene” refers to an unsaturated, branched or straightchain or cyclic hydrocarbon radical of 2-18 carbon atoms, and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkyne. Typicalalkynylene radicals include, but are not limited to: acetylene (—C≡C—),propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡CH—).

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Some aryl groups are representedin the exemplary structures as “Ar”. Typical aryl groups include, butare not limited to, radicals derived from benzene, substituted benzene,naphthalene, anthracene, biphenyl, and the like.

“Arylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl radical. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. The arylalkyl group comprises 6 to 20 carbon atoms, e.g., thealkyl moiety, including alkanyl, alkenyl or alkynyl groups, of thearylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14carbon atoms.

“Heteroarylalkyl” refers to an acyclic alkyl radical in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with a heteroaryl radical. Typicalheteroarylalkyl groups include, but are not limited to,2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroarylalkylgroup comprises 6 to 20 carbon atoms, e.g., the alkyl moiety, includingalkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety ofthe heteroarylalkyl group may be a monocycle having 3 to 7 ring members(2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), forexample: a bicyclo [4,5], [5,5], [5,6], or [6,6] system. “Substitutedalkyl”, “substituted aryl”, and “substituted arylalkyl” mean alkyl,aryl, and arylalkyl respectively, in which one or more hydrogen atomsare each independently replaced with a substituent. Typical substituentsinclude, but are not limited to, —X, —R, —O⁻, —OR, —SR, —S⁻, —NR₂, —NR₃,═NR, —CX₃, —CN, —OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, NC(═O)R,—C(═O)R, —C(═O)NR₂, —SO₃ ⁻, —SO₃H, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR,—S(═O)R, —OP(═O)(OR)₂, —P(═O)(OR)₂, —PO⁻ ₃, —PO₃H₂, —C(═O)R, —C(═O)X,—C(═S)R, —CO₂R, —CO₂ ⁻, —C(═S)OR, —C(═O)SR, —C(═S)SR, —C(═O)NR₂,—C(═S)NR₂, —C(═NR)NR₂, where each X is independently a halogen: F, Cl,Br, or I; and each R is independently —H, C₂-C₁₈ alkyl, C₆-C₂₀ aryl,C₃-C₁₄ heterocycle, protecting group or prodrug moiety. Alkylene,alkenylene, and alkynylene groups as described above may also besimilarly substituted.

“Heteroaryl” and “Heterocycle” refer to a ring system in which one ormore ring atoms is a heteroatom, e.g., nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 1 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo [4,5], [5,5], [5,6], or [6,6]system.

Heterocycles are described in Paquette, Leo A.; “Principles of ModernHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.(1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

“Carbocycle” means a saturated or unsaturated ring having 3 to 7 carbonatoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Monocycliccarbocycles have 3 to 6 ring atoms, still more typically 5 or 6 ringatoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g., arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. Examples of monocycliccarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

“Linker”, “Linker Unit”, or “link” means a chemical moiety comprising acovalent bond or a chain of atoms that covalently attaches an antibodyto a drug moiety. In various embodiments, a linker is specified as LU.Linkers include a divalent radical such as an alkyldiyl, an aryldiyl, aheteroaryldiyl, moieties such as: —(CR₂)_(n)O(CR₂)_(n)—, repeating unitsof alkyloxy (e.g., polyethylenoxy, PEG, polymethyleneoxy) and alkylamino(e.g., polyethyleneamino, Jeffamine™); and diacid ester and amidesincluding succinate, succinamide, diglycolate, malonate, and caproamide.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g., melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and l or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or l meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

Examples of a “patient” include, but are not limited to, a human, rat,mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird andfowl. In an exemplary embodiment, the patient is a human.

“Aryl” refers to a carbocyclic aromatic group. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracenyl. Acarbocyclic aromatic group or a heterocyclic aromatic group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′,—OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

The term “C₁-C₈ alkyl,” as used herein refers to a straight chain orbranched, saturated or unsaturated hydrocarbon having from 1 to 8 carbonatoms. Representative “C₁-C₈ alkyl” groups include, but are not limitedto, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl,-n-heptyl, -n-octyl, -n-nonyl and -n-decyl; while branched C₁-C₈ alkylsinclude, but are not limited to, -isopropyl, -sec-butyl, -isobutyl,-tert-butyl, -isopentyl, 2-methylbutyl, unsaturated C₁-C₈ alkylsinclude, but are not limited to, -vinyl, -allyl, -1-butenyl, -2-butenyl,-isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl,-2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, 1-hexyl, 2-hexyl, 3-hexyl,-acetylenyl, -propynyl, -1-butynyl, -2-butynyl, -1-pentynyl,-2-pentynyl, -3-methyl-1 butynyl. methyl, ethyl, propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl,neopentyl, n-hexyl, isohexyl, 2-methylpentyl, 3-methylpentyl,2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl,2,3-dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl,3-methylhexyl, 2,2-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl,3,5-dimethylhexyl, 2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl,n-heptyl, isoheptyl, n-octyl, and isooctyl. A C₁-C₈ alkyl group can beunsubstituted or substituted with one or more groups including, but notlimited to, —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, -halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where eachR′ is independently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocycle” is a 3-, 4-, 5-, 6-, 7- or 8-membered saturated orunsaturated non-aromatic carbocyclic ring. Representative C₃-C₈carbocycles include, but are not limited to, -cyclopropyl, -cyclobutyl,-cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl,-1,3-cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl,-1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl, -cyclooctyl, and-cyclooctadienyl. A C₃-C₈ carbocycle group can be unsubstituted orsubstituted with one or more groups including, but not limited to,—C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH,-halogen, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN; where each R′ isindependently selected from H, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ carbocyclo” refers to a C₃-C₈ carbocycle group defined abovewherein one of the carbocycle groups' hydrogen atoms is replaced with abond.

A “C₁-C₁₀ alkylene” is a straight chain, saturated hydrocarbon group ofthe formula —(CH₂)₁₋₁₀₋. Examples of a C₁-C₁₀ alkylene includemethylene, ethylene, propylene, butylene, pentylene, hexylene,heptylene, ocytylene, nonylene and decalene.

An “arylene” is an aryl group which has two covalent bonds and can be inthe ortho, meta, or para configurations as shown in the followingstructures:

in which the phenyl group can be unsubstituted or substituted with up tofour groups including, but not limited to, —C₃-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

A “C₃-C₈ heterocycle” refers to an aromatic or non-aromatic C₃-C₈carbocycle in which one to four of the ring carbon atoms areindependently replaced with a heteroatom from the group consisting of O,S and N. Representative examples of a C₃-C₈ heterocycle include, but arenot limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl. A C₃-C₈ heterocycle can be unsubstituted or substituted withup to seven groups including, but not limited to, —C₁-C₈ alkyl,—O—(C₁-C₈ alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃,—NH₂, —NH(R′), —N(R′)₂ and —CN; wherein each R′ is independentlyselected from H, —C₁-C₈ alkyl and aryl.

“C₃-C₈ heterocyclo” refers to a C₃-C₈ heterocycle group defined abovewherein one of the heterocycle group's hydrogen atoms is replaced with abond. A C₃-C₈ heterocyclo can be unsubstituted or substituted with up tosix groups including, but not limited to, —C₁-C₈ alkyl, —O—(C₁-C₈alkyl), -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′,—C(O)N(R′)₂—NHC(O)R′, —S(O)₂R′, —S(O)R′, —OH, -halogen, —N₃, —NH₂,—NH(R′), —N(R′)₂ and —CN; wherein each R′ is independently selected fromH, —C₁-C₈ alkyl and aryl.

An “Exemplary Compound” is a Drug Compound or a Drug-Linker Compound.

An “Exemplary Conjugate” is a Drug-Ligand Conjugate having a cleavableDrug unit from the Drug-Ligand Conjugate or a Drug-Linker-LigandConjugate.

In some embodiments, the Exemplary Compounds and Exemplary Conjugatesare in isolated or purified form. As used herein, “isolated” meansseparated from other components of (a) a natural source, such as a plantor animal cell or cell culture, or (b) a synthetic organic chemicalreaction mixture. As used herein, “purified” means that when isolated,the isolate contains at least 95%, and in another aspect at least 98%,of Exemplary Compound or Exemplary Conjugate by weight of the isolate.

Examples of a “hydroxyl protecting group” include, but are not limitedto, methoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranylether, benzyl ether, p-methoxybenzyl ether, trimethylsilyl ether,triethylsilyl ether, triisopropyl silyl ether, t-butyldimethyl silylether, triphenylmethyl silyl ether, acetate ester, substituted acetateesters, pivaloate, benzoate, methanesulfonate and p-toluenesulfonate.

“Leaving group” refers to a functional group that can be substituted byanother functional group. Such leaving groups are well known in the art,and examples include, but are not limited to, a halide (e.g., chloride,bromide, iodide), methanesulfonyl (mesyl), p-toluenesulfonyl (tosyl),trifluoromethylsulfonyl (triflate), and trifluoromethylsulfonate. Thephrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of an ExemplaryCompound or Exemplary Conjugate.

The Exemplary Compounds and Exemplary Conjugates contain at least oneamino group, and accordingly acid addition salts can be formed with thisamino group. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counter ions. Hence, apharmaceutically acceptable salt can have one or more charged atomsand/or one or more counterion.

“Pharmaceutically acceptable solvate” or “solvate” refer to anassociation of one or more solvent molecules and a compound of theinvention, e.g., an Exemplary Compound or Exemplary Conjugate. Examplesof solvents that form pharmaceutically acceptable solvates include, butare not limited to, water, isopropanol, ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine.

The following abbreviations are used herein and have the indicateddefinitions: AE is auristatin E, Boc is N-(t-butoxycarbonyl), cit iscitrulline, dap is dolaproine, DCC is 1,3-dicyclohexylcarbodiimide, DCMis dichloromethane, DEA is diethylamine, DEAD isdiethylazodicarboxylate, DEPC is diethylphosphorylcyanidate, DIAD isdiisopropylazodicarboxylate, DIEA is N,N-diisopropylethylamine, dil isdolaisoleuine, DMAP is 4-dimethylaminopyridine, DME is ethyleneglycoldimethyl ether (or 1,2-dimethoxyethane), DMF is N,N-dimethylformamide,DMSO is dimethylsulfoxide, doe is dolaphenine, dov isN,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid), DTPA isdiethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCI is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine,MeCN(CH₃CN) is acetonitrile, MeOH is methanol, Mtr is4-anisyldiphenylmethyl (or 4-methoxytrityl), nor is (1S,2R)-(+)-norephedrine, PAB is p-aminobenzyl, PBS is phosphate-bufferedsaline (pH 7.4), PEG is polyethylene glycol, Ph is phenyl, Pnp isp-nitrophenyl, MC is 6-maleimidocaproyl, phe is L-phenylalanine, PyBropis bromo tris-pyrrolidino phosphonium hexafluorophosphate, SEC issize-exclusion chromatography, Su is succinimide, TBTU isO-benzotriazol-1-yl-N,N,N,N-tetramethyluronium tetrafluoroborate, TFA istrifluoroacetic acid, TLC is thin layer chromatography, UV isultraviolet, and val is valine.

The following linker abbreviations are used herein and have theindicated definitions: Val Cit is a valine-citrulline, dipeptide site inprotease cleavable linker; PAB is p-aminobenzylcarbamoyl; (Me)vc isN-methyl-valine citrulline, where the linker peptide bond has beenmodified to prevent its cleavage by cathepsin B; MC(PEG)6-OH ismaleimidocaproyl- polyethylene glycol; SPP is N-Succinimidyl4-(2-pyridylthio) pentanoate; and SMCC is N-Succinimidyl4-(N-maleimidomethyl)cyclohexane-1 carboxylate.

The terms “treat” or “treatment,” unless otherwise indicated by context,refer to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) anundesired physiological change or disorder, such as the development orspread of cancer. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder or those in which the condition or disorder is tobe prevented.

In the context of cancer, the term “treating” includes any or all of:preventing growth of tumor cells, cancer cells, or of a tumor;preventing replication of tumor cells or cancer cells, lessening ofoverall tumor burden or decreasing the number of cancerous cells, andameliorating one or more symptoms associated with the disease.

In the context of an autoimmune disease, the term “treating” includesany or all of: preventing replication of cells associated with anautoimmune disease state including, but not limited to, cells thatproduce an autoimmune antibody, lessening the autoimmune-antibody burdenand ameliorating one or more symptoms of an autoimmune disease.

In the context of an infectious disease, the term “treating” includesany or all of: preventing the growth, multiplication or replication ofthe pathogen that causes the infectious disease and ameliorating one ormore symptoms of an infectious disease.

The following cytotoxic drug abbreviations are used herein and have theindicated definitions: MMAE is mono-methyl auristatin E (MW 718); MMAFis N-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine (MW731.5); MMAF-DMAEA is MMAF with DMAEA (dimethylaminoethylamine) in anamide linkage to the C-terminal phenylalanine (MW 801.5); MMAF-TEG isMMAF with tetraethylene glycol esterified to the phenylalanine;MMAF-NtBu is N-t-butyl, attached as an amide to C-terminus of MMAF; AEVBis auristatin E valeryl benzylhydrazone, acid labile linker through theC-terminus of AE (MW 732); and AFP is Monoamide of p-phenylene diaminewith C-terminal Phenylalanine of Auristatin F (MW 732).

The Compounds of the Invention

The Compounds of Formula (Ia)

In one aspect, the invention provides Drug-Linker-Ligand Conjugateshaving Formula Ia:

LA_(a)-W_(w)—Y_(y)-D)_(p)  Ia

-   -   or a pharmaceutically acceptable salt or solvate thereof    -   wherein,    -   L- is a Ligand unit;    -   -A_(a)-W_(w)—Y_(y)— is a Linker unit (LU), wherein the Linker        unit includes:    -   -A- is a Stretcher unit,    -   a is 0 or 1,    -   each —W— is independently an Amino Acid unit,    -   w is an integer ranging from 0 to 12,    -   —Y— is a Spacer unit, and    -   y is 0, 1 or 2;    -   p ranges from 1 to about 20; and    -   -D is a Drug unit having the Formulas D_(E) and D_(F):

-   -   wherein, independently at each location:    -   R² is selected from H and C₁-C₈ alkyl;    -   R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁵ is selected from H and methyl;    -   or R⁴ and R⁵ jointly form a carbocyclic ring and have the        formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from H, C₁-C₈ alkyl and C₃-C₈ carbocycle        and n is selected from 2, 3, 4, 5 and 6;    -   R⁶ is selected from H and C₁-C₈ alkyl;    -   R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈        carbocycle and O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and C₁-C₈ alkyl;    -   R¹⁰ is selected from aryl or C₃-C₈ heterocycle;    -   Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is C₂-C₈ alkyl;    -   R¹⁴ is H or C₁-C₈ alkyl;    -   each occurrence of R¹⁵ is independently H, COOH,        —(CH₂)_(n)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈        alkyl;    -   each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH;    -   R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈        heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and    -   n is an integer ranging from 0 to 6.

In another embodiment, the present invention provides Drug Compoundshaving the Formula Ib:

or pharmaceutically acceptable salts or solvates thereof,

-   -   wherein:    -   R² is selected from hydrogen and —C₁-C₈ alkyl;    -   R³ is selected from hydrogen, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈        heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from hydrogen, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle),        —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle) wherein        R⁵ is selected from —H and -methyl; or R⁴ and R⁵ jointly, have        the formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the carbon atom to which they are attached;    -   R⁶ is selected from H and —C₁-C₈ alkyl;    -   R⁷ is selected from H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, aryl,        —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈        heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from aryl group or —C₃-C₈ heterocycle;    -   Z is —O—, —S—, —NH—, or —NR¹²—, wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, —C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is —C₂-C₈ alkyl;    -   R¹⁴ is H or —C₁-C₈ alkyl;    -   each occurrence of R¹⁵ is independently H, —COOH,        —(CH₂)_(n)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈        alkyl;    -   each occurrence of R¹⁶ is independently H, —C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH; and    -   n is an integer ranging from 0 to 6.

In yet another embodiment, the invention provides Drug-Linker-LigandConjugates having the Formula Ia′:

AbA _(a)-W_(w)—Y_(y)-D)_(p)  Formula Ia′

or pharmaceutically acceptable salts or solvates thereof.

-   -   wherein:    -   Ab is an antibody,    -   A is a Stretcher unit,    -   a is 0 or 1,    -   each W is independently an Amino Acid unit,    -   w is an integer ranging from 0 to 12,    -   Y is a Spacer unit, and    -   y is 0, 1 or 2,    -   p ranges from 1 to about 20, and    -   D is a Drug moiety selected from Formulas D_(E) and IX:

-   -   wherein, independently at each location:    -   R² is selected from H and C₁-C₈ alkyl;    -   R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁵ is selected from H and methyl;    -   or R⁴ and R⁵ jointly form a carbocyclic ring and have the        formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from H, C₁-C₈ alkyl and C₃-C₈ carbocycle        and n is selected from 2, 3, 4, 5 and 6;    -   R⁶ is selected from H and C₁-C₈ alkyl;    -   R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈        carbocycle and O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and C₁-C₈ alkyl;    -   R¹⁰ is selected from aryl or C₃-C₈ heterocycle;    -   Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is C₂-C₈ alkyl;    -   R¹⁴ is H or C₁-C₈ alkyl;    -   each occurrence of R¹⁵ is independently H, COON, —(CH₂),        —N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;    -   each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH;    -   R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈        heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and    -   n is an integer ranging from 0 to 6.    -   Ab is any antibody covalently attached to one or more drug        units. Ab includes an antibody which binds to CD30, CD40, CD70,        Lewis Y antigen. In another embodiment, Ab does not include an        antibody which binds to an ErbB receptor or to one or more of        receptors    -   (1)-(35):    -   (1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank        accession no. NM_(—)001203);    -   (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_(—)003486);    -   (3) STEAP1 (six transmembrane epithelial antigen of prostate,        Genbank accession no. NM_(—)012449);    -   (4) 0772P (CA125, MUC16, Genbank accession no. AF361486);    -   (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,        mesothelin, Genbank accession no. NM_(—)005823);    -   (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34        (sodium phosphate), member 2, type II sodium-dependent phosphate        transporter 3b, Genbank accession no. NM_(—)006424);    -   (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,        Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats        (type 1 and type 1-like), transmembrane domain (TM) and short        cytoplasmic domain, (semaphorin) 5B, Genbank accession no.        AB040878);    -   (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA        2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no.        AY358628);    -   (9) ETBR (Endothelin type B receptor, Genbank accession no.        AY275463);    -   (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank        accession no. NM_(—)017763);    -   (11) STEAP2 (HGNC_(—)8639, IPCA-1, PCANAP1, STAMP1, STEAP2,        STMP, prostate cancer associated gene 1, prostate cancer        associated protein 1, six transmembrane epithelial antigen of        prostate 2, six transmembrane prostate protein, Genbank        accession no. AF455138);    -   (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor        potential cation channel, subfamily M, member 4, Genbank        accession no. NM_(—)017636);    -   (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,        teratocarcinoma-derived growth factor, Genbank accession no.        NP_(—)003203 or NM_(—)003212);    -   (14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr        virus receptor) or Hs.73792, Genbank accession no. M26004);    -   (15) CD79b (1 Gb (immunoglobulin-associated beta), B29, Genbank        accession no. NM_(—)000626);    -   (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing        phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank        accession no. NM_(—)030764);    -   (17) HER2 (Genbank accession no. M11730);    -   (18) NCA (Genbank accession no. M18728);    -   (19) MDP (Genbank accession no. BC017023);    -   (20) IL20Rα (Genbank accession no. AF184971);    -   (21) Brevican (Genbank accession no. AF229053);    -   (22) Ephb2R (Genbank accession no. NM_(—)004442);    -   (23) ASLG659 (Genbank accession no. AX092328);    -   (24) PSCA (Genbank accession no. AJ297436);    -   (25) GEDA (Genbank accession no. AY260763);    -   (26) BAFF-R (Genbank accession no. NP_(—)443177.1);    -   (27) CD22 (Genbank accession no. NP-001762.1);    -   (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a B        cell-specific protein that covalently interacts with Ig beta        (CD79B) and forms a complex on the surface with Ig M molecules,        transduces a signal involved in B-cell differentiation, Genbank        accession No. NP_(—)001774.1);    -   (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled        receptor that is activated by the CXCL13 chemokine, functions in        lymphocyte migration and humoral defense, plays a role in HIV-2        infection and perhaps development of AIDS, lymphoma, myeloma,        and leukemia, Genbank accession No. NP_(—)001707.1);    -   (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)        that binds peptides and presents them to CD4+ T lymphocytes,        Genbank accession No. NP_(—)002111.1);    -   (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5,        an ion channel gated by extracellular ATP, may be involved in        synaptic transmission and neurogenesis, deficiency may        contribute to the pathophysiology of idiopathic detrusor        instability, Genbank accession No. NP_(—)002552.2);    -   (32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank        accession No. NP_(—)001773.1);    -   (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane        protein of the leucine rich repeat (LRR) family, regulates        B-cell activation and apoptosis, loss of function is associated        with increased disease activity in patients with systemic lupus        erythematosis, Genbank accession No. NP_(—)005573.1);    -   (34) FCRH1 (Fc receptor-like protein 1, a putative receptor for        the immunoglobulin Fc domain that contains C2 type Ig-like and        ITAM domains, may have a role in B-lymphocyte differentiation,        Genbank accession No. NP_(—)443170.1); and/or    -   (35) IRTA2 (Immunoglobulin superfamily receptor translocation        associated 2, a putative immunoreceptor with possible roles in B        cell development and lymphomagenesis; deregulation of the gene        by translocation occurs in some B cell malignancies, Genbank        accession No. NP_(—)112571.1).

In one embodiment —Ww- is -Val-Cit-.

In another embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is —H, andR⁷ is sec-butyl. In yet another embodiment, R² and R⁶ are each methyl,and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

In one aspect, Ab is cAC10, cBR96, cS2C6, c1F6, c2F2, hAC10, hBR96,hS2C6, h1F6, and h2F2.

Exemplary embodiments of Formula Ia have the following structures:

wherein L is an antibody, Val is valine, and Cit is citrulline.

The drug loading is represented by p, the average number of drugmolecules per antibody in a molecule (e.g., of Formula Ia, Ia′ and Ic).Drug loading may range from 1 to 20 drugs (D) per Ligand (e.g., Ab ormAb). Compositions of Formula Ia and Formula Ia′ include collections ofantibodies conjugated with a range of drugs, from 1 to 20. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution of Ligand-Drug-Conjugatesin terms of p may also be determined. In some instances, separation,purification, and characterization of homogeneous Ligand-Drug-conjugateswhere p is a certain value from Ligand-Drug-Conjugates with other drugloadings may be achieved by means such as reverse phase HPLC orelectrophoresis.

The Drug Compounds of Formula (Ib)

In another aspect, the present invention provides Drug Compounds havingthe Formula (Ib):

-   -   or a pharmaceutically acceptable salt or solvate thereof,    -   wherein:    -   R² is selected from -hydrogen and —C₁-C₈ alkyl;    -   R³ is selected from -hydrogen, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        aryl, —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈        heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from -hydrogen, —C₁-C₈ alkyl, —C₃-C₈ carbocycle,        -aryl, —C₁-C₈ alkyl-aryl, —C₁-C₃ alkyl-(C₃-C₈ carbocycle),        —C₃-C₈ heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle) wherein        R⁵ is selected from and -methyl; or R⁴ and R⁵ jointly, have the        formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈        carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a        ring with the carbon atom to which they are attached;    -   R⁶ is selected from —H and —C₁-C₈ alkyl;    -   R⁷ is selected from —H, —C₁-C₈ alkyl, —C₃-C₈ carbocycle, aryl,        —C₁-C₈ alkyl-aryl, —C₁-C₈ alkyl-(C₃-C₈ carbocycle), —C₃-C₈        heterocycle and —C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from —H, —OH, —C₁-C₈ alkyl,        —C₃-C₈ carbocycle and —O—(C₁-C₈ alkyl);    -   R⁹ is selected from —H and —C₁-C₈ alkyl;    -   R¹⁰ is selected from aryl group or —C₃-C₈ heterocycle;    -   Z is —O—, —S—, —NH—, or —NR¹²—, wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from —H, C₁-C₂₀ alkyl, aryl, —C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is —C₂-C₈ alkyl;    -   R¹⁴ is —H or —C₁-C₈ alkyl; each occurrence of R¹⁵ is        independently —H, —COOH, —(CH₂)_(m)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or        —(CH₂)_(n)—SO₃—C₁-C₈ alkyl;    -   each occurrence of R¹⁶ is independently —H, —C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH; and    -   n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H. In an exemplary embodiment, R³ and R⁴ are eachisopropyl, R⁵ is —H, and R⁷ is sec-butyl.

In another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

Illustrative Compounds of Formula (Ib), each of which may be used asdrug moieties (D) in ADC, include compounds having the followingstructures:

and pharmaceutically acceptable salts or solvates thereof.

The Compounds of Formula (Ic)

In another aspect, the invention provides antibody-drug conjugatecompounds (ADC) having Formula Ic:

AbA _(a)-W_(w)—Y_(y)-D)_(p)  Ic

comprising an antibody covalently attached to one or more drug units(moieites). The antibody-drug conjugate compounds includepharmaceutically acceptable salts or solvates thereof.

Formula Ic compounds are defined wherein:

-   -   Ab is an antibody which binds to one or more tumor-associated        antigen receptors (1)-(35):    -   (1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbank        accession no. NM_(—)001203);    -   (2) E16 (LAT1, SLC7A5, Genbank accession no. NM_(—)003486);    -   (3) STEAP1 (six transmembrane epithelial antigen of prostate,        Genbank accession no. NM_(—)012449);    -   (4) 0772P (CA125, MUC16, Genbank accession no. AF361486);    -   (5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,        mesothelin, Genbank accession no. NM_(—)005823);    -   (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34        (sodium phosphate), member 2, type II sodium-dependent phosphate        transporter 3b, Genbank accession no. NM_(—)006424);    -   (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG,        Semaphorin 5b Hlog, sema domain, seven thrombospondin repeats        (type 1 and type 1-like), transmembrane domain (TM) and short        cytoplasmic domain, (semaphorin) 5B, Genbank accession no.        AB040878);    -   (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA        2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession no.        AY358628);    -   (9) ETBR (Endothelin type B receptor, Genbank accession no.        AY275463);    -   (10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank        accession no. NM_(—)017763);    -   (11) STEAP2 (HGNC_(—)8639, IPCA-1, PCANAP1, STAMP1, STEAP2,        STMP, prostate cancer associated gene 1, prostate cancer        associated protein 1, six transmembrane epithelial antigen of        prostate 2, six transmembrane prostate protein, Genbank        accession no. AF455138);    -   (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor        potential cation channel, subfamily M, member 4, Genbank        accession no. NM_(—)017636);    -   (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,        teratocarcinoma-derived growth factor, Genbank accession no.        NP_(—)003203 or NM_(—)003212);    -   (14) CD21 (CR2 (Complement receptor 2) or C3DR(C3d/Epstein Barr        virus receptor) or Hs.73792 Genbank accession no. M26004);    -   (15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta),        B29, Genbank accession no. NM_(—)000626);    -   (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing        phosphatase anchor protein 1a), SPAP1B, SPAP1C, Genbank        accession no. NM_(—)030764);    -   (17) HER2 (Genbank accession no. M11730);    -   (18) NCA (Genbank accession no. M18728);    -   (19) MDP (Genbank accession no. BC017023);    -   (20) IL20Rα (Genbank accession no. AF184971);    -   (21) Brevican (Genbank accession no. AF229053);    -   (22) Ephb2R (Genbank accession no. NM_(—)004442);    -   (23) ASLG659 (Genbank accession no. AX092328);    -   (24) PSCA (Genbank accession no. AJ297436);    -   (25) GEDA (Genbank accession no. AY260763;    -   (26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3,        BR3, NP_(—)443177.1);    -   (27) CD22 (B-cell receptor CD22-B isoform, NP-001762.1);    -   (28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a B        cell-specific protein that covalently interacts with Ig beta        (CD79B) and forms a complex on the surface with Ig M molecules,        transduces a signal involved in B-cell differentiation, Genbank        accession No. NP_(—)001774.1);    -   (29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled        receptor that is activated by the CXCL13 chemokine, functions in        lymphocyte migration and humoral defense, plays a role in HIV-2        infection and perhaps development of AIDS, lymphoma, myeloma,        and leukemia, Genbank accession No. NP_(—)001707.1);    -   (30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen)        that binds peptides and presents them to CD4+ T lymphocytes,        Genbank accession No. NP_(—)002111.1);    -   (31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5,        an ion channel gated by extracellular ATP, may be involved in        synaptic transmission and neurogenesis, deficiency may        contribute to the pathophysiology of idiopathic detrusor        instability, Genbank accession No. NP_(—)002552.2);    -   (32) CD72 (B-cell differentiation antigen CD72, Lyb-2, Genbank        accession No. NP_(—)001773.1);    -   (33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane        protein of the leucine rich repeat (LRR) family, regulates        B-cell activation and apoptosis, loss of function is associated        with increased disease activity in patients with systemic lupus        erythematosis, Genbank accession No. NP_(—)005573.1);    -   (34) FCRH1 (Fc receptor-like protein 1, a putative receptor for        the immunoglobulin Fc domain that contains C2 type Ig-like and        ITAM domains, may have a role in B-lymphocyte differentiation,        Genbank accession No. NP_(—)443170.1); and    -   (35) IRTA2 (Immunoglobulin superfamily receptor translocation        associated 2, a putative immunoreceptor with possible roles in B        cell development and lymphomagenesis; deregulation of the gene        by translocation occurs in some B cell malignancies, Genbank        accession No. NP_(—)112571.1).    -   A is a Stretcher unit,    -   a is 0 or 1,    -   each W is independently an Amino Acid unit,    -   w is an integer ranging from 0 to 12,    -   Y is a Spacer unit, and    -   y is 0, 1 or 2,    -   p ranges from 1 to about 8, and    -   D is a Drug moiety selected from Formulas D_(E) and D_(F):

wherein the wavy line of D_(E) and D_(F) indicates the covalentattachment site to A, W, or Y, and independently at each location:

-   -   R² is selected from H and C₁-C₈ alkyl;    -   R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁵ is selected from H and methyl;    -   or R⁴ and R⁵ jointly form a carbocyclic ring and have the        formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from H, C₁-C₈ alkyl and C₃-C₈ carbocycle        and n is selected from 2, 3, 4, 5 and 6;    -   R⁶ is selected from H and C₁-C₈ alkyl;    -   R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈        carbocycle and O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and C₁-C₈ alkyl;    -   R¹⁰ is selected from aryl or C₃-C₈ heterocycle;    -   Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is C₂-C₈ alkyl;    -   R¹⁴ is H or C₁-C₈ alkyl;    -   each occurrence of R¹⁵ is independently H, COOH,        —(CH₂)_(n)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈        alkyl;    -   each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH;    -   R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈        heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and    -   n is an integer ranging from 0 to 6.

In one embodiment —Ww- is -Val-Cit-.

In another embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is —H, andR⁷ is sec-butyl.

In yet another embodiment, R² and R⁶ are each methyl, and R⁹ is —H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is —H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is —H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl.

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is —H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H.

Exemplary embodiments of Formula Ic ADC have the following structures:

wherein Ab is an antibody which binds to one or more tumor-associatedantigen receptors (1)-(35); Val is valine; and Cit is citrulline.

The drug loading is represented by p, the average number of drugs perantibody in a molecule of Formula I. Drug loading may range from 1 to 20drugs (D) per antibody (Ab or mAb). Compositions of ADC of Formula Iinclude collections of antibodies conjugated with a range of drugs, from1 to 20. The average number of drugs per antibody in preparations of ADCfrom conjugation reactions may be characterized by conventional meanssuch as UV/visible spectroscopy, mass spectrometry, ELISA assay, andHPLC. The quantitative distribution of ADC in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADC where p is a certain value from ADCwith other drug loadings may be achieved by means such as reverse phaseHPLC or electrophoresis.

For some antibody drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached.

Typically, fewer than the theoretical maximum of drug moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with thedrug-linker intermediate or linker reagent. Only the most reactivelysine groups may react with an amine-reactive linker reagent.Generally, antibodies do not contain many, if any, free and reactivecysteine thiol groups which may be linked to a drug moiety. Mostcysteine thiol residues in the antibodies of the compounds of theinvention exist as disulfide bridges and must be reduced with a reducingagent such as dithiothreitol (DTT). Additionally, the antibody must besubjected to denaturing conditions to reveal reactive nucleophilicgroups such as lysine or cysteine. The loading (drug/antibody ratio) ofan ADC may be controlled in several different manners, including: (i)limiting the molar excess of drug-linker intermediate or linker reagentrelative to antibody, (ii) limiting the conjugation reaction time ortemperature, and (iii) partial or limiting reductive conditions forcysteine thiol modification.

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate, or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by dual ELISA antibody assay, specific for antibody andspecific for the drug. Individual ADC molecules may be identified in themixture by mass spectroscopy, and separated by HPLC, e.g., hydrophobicinteraction chromatography (“Effect of drug loading on the pharmacology,pharmacokinetics, and toxicity of an anti-CD30 antibody-drug conjugate”,Hamblett, K. J., et al, Abstract No. 624, American Association forCancer Research; 2004 Annual Meeting, Mar. 27-31, 2004, Proceedings ofthe AACR, Volume 45, March 2004; “Controlling the Location of DrugAttachment in Antibody-Drug Conjugates”, Alley, S. C., et al, AbstractNo. 627, American Association for Cancer Research; 2004 Annual Meeting,Mar. 27-31, 2004, Proceedings of the AACR, Volume 45, March 2004). Thus,a homogeneous ADC with a single loading value may be isolated from theconjugation mixture by electrophoresis or chromatography.

The Linker Unit

A “Linker unit” (LU) is a bifunctional compound which can be used tolink a Drug unit and an Ligand unit to form Drug-Linker-LigandConjugates, or which are useful in the formation of immunoconjugatesdirected against tumor associated antigens. Such immunoconjugates allowthe selective delivery of toxic drugs to tumor cells.

In one embodiment, the Linker unit of the Drug-Linker Compound andDrug-Linker-Ligand Conjugate has the formula:

-A_(a)-W_(w)—Y_(y)—

wherein:

-   -   -A- is a Stretcher unit;    -   a is 0 or 1;    -   each —W— is independently an Amino Acid unit;    -   w is independently an integer ranging from 0 to 12;    -   —Y— is a Spacer unit; and    -   y is 0, 1 or 2.

In the Drug-Linker-Ligand Conjugate, the Linker is capable of linkingthe Drug moiety and the Ligand unit.

The Stretcher Unit

The Stretcher unit (-A-), when present, is capable of linking a Ligandunit to an amino acid unit (—W—). In this regard a Ligand (L) has afunctional group that can form a bond with a functional group of aStretcher. Useful functional groups that can be present on a ligand,either naturally or via chemical manipulation include, but are notlimited to, sulfhydryl (—SH), amino, hydroxyl, carboxy, the anomerichydroxyl group of a carbohydrate, and carboxyl. In one aspect, theLigand functional groups are sulfhydryl and amino. Sulfhydryl groups canbe generated by reduction of an intramolecular disulfide bond of aLigand. Alternatively, sulfhydryl groups can be generated by reaction ofan amino group of a lysine moiety of a Ligand using 2-iminothiolane(Traut's reagent) or another sulfhydryl generating reagent.

In one embodiment, the Stretcher unit forms a bond with a sulfur atom ofthe Ligand unit. The sulfur atom can be derived from a sulfhydryl groupof a Ligand. Representative Stretcher units of this embodiment aredepicted within the square brackets of Formulas IIIa and IIIb, whereinL-, —W—, —Y—, -D, w and y are as defined above, and R¹⁷ is selected from—C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-, —O—(C₁-C₈ alkyl)-, -arylene-,—C₁-C₁₀ alkylene-arylene-, -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈ carbocyclo)-C₁-C₁₀ alkylene-,—C₃-C₈ heterocyclo-, —C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈heterocyclo)-C₁-C₁₀ alkylene-, —(CH₂CH₂O)_(r)—, and —(CH₂CH₂O)_(r)—CH₂—;and r is an integer ranging from 1-10. It is to be understood from allthe exemplary embodiments of Formula Ia, such as III-VI, that even wherenot denoted expressly, from 1 to 20 drug moieties are linked to a Ligand(p=1-20).

An illustrative Stretcher unit is that of Formula IIIc wherein R¹⁷ is—(CH₂)₅—:

Another illustrative Stretcher unit is that of Formula IIIa wherein R¹⁷is —(CH₂CH₂O)_(r)—CH₂—; and r is 2:

Still another illustrative Stretcher unit is that of Formula IIIbwherein R¹⁷ is —(CH₂)₅—:

In another embodiment, the Stretcher unit is linked to the Ligand unitvia a disulfide bond between a sulfur atom of the Ligand unit and asulfur atom of the Stretcher unit. A representative Stretcher unit ofthis embodiment is depicted within the square brackets of Formula IV,wherein R¹⁷, L-, —W—, —Y—, -D, w and y are as defined above.

LS—R¹⁷—C(O)W_(w)—Y_(y)-D  IV

In yet another embodiment, the reactive group of the Stretcher containsa reactive site that can form a bond with a primary or secondary aminogroup of a Ligand. Example of these reactive sites include, but are notlimited to, activated esters such as succinimide esters, 4-nitrophenylesters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides,acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative Stretcher units of this embodiment are depicted withinthe square brackets of Formulas Va and Vb, wherein —R¹⁷—, L-, —W—, —Y—,-D, w and y are as defined above;

In yet another aspect, the reactive group of the Stretcher contains areactive site that is reactive to a modified carbohydrate's (—CHO) groupthat can be present on a Ligand. For example, a carbohydrate can bemildly oxidized using a reagent such as sodium periodate and theresulting (—CHO) unit of the oxidized carbohydrate can be condensed witha Stretcher that contains a functionality such as a hydrazide, an oxime,a primary or secondary amine, a hydrazine, a thiosemicarbazone, ahydrazine carboxylate, and an arylhydrazide such as those described byKaneko, T. et al. (1991) Bioconjugate Chem 2:133-41. RepresentativeStretcher units of this embodiment are depicted within the squarebrackets of Formulas VIa, VIb, and VIc, wherein —R¹⁷—, L-, —W—, —Y—, -D,w and y are as defined above.

The Amino Acid Unit

The Amino Acid unit (—W—), when present, links the Stretcher unit to theSpacer unit if the Spacer unit is present, links the Stretcher unit tothe Drug moiety if the Spacer unit is absent, and links the Ligand unitto the Drug unit if the Stretcher unit and Spacer unit are absent.

W_(w)— is a dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit. Each —W— unit independently has theformula denoted below in the square brackets, and w is an integerranging from 0 to 12:

wherein R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

The Amino Acid unit can be enzymatically cleaved by one or more enzymes,including a tumor-associated protease, to liberate the Drug unit (-D),which in one embodiment is protonated in vivo upon release to provide aDrug (D).

Illustrative W_(w) units are represented by formulas (VII)-(IX):

wherein R²⁰ and R²¹ are as follows:

R²⁰ R²¹ benzyl (CH₂)₄NH₂; methyl (CH₂)₄NH₂; isopropyl (CH₂)₄NH₂;isopropyl (CH₂)₃NHCONH₂; benzyl (CH₂)₃NHCONH₂; isobutyl (CH₂)₃NHCONH₂;sec-butyl (CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂; benzyl methyl; and benzyl (CH₂)₃NHC(═NH)NH₂;

wherein R²⁰, R²¹ and R²² are as follows:

R²⁰ R²¹ R²² benzyl Benzyl (CH₂)₄NH₂; isopropyl Benzyl (CH₂)₄NH₂; and HBenzyl (CH₂)₄NH₂;

wherein R²⁰, R²¹, R²² and R²³ are as follows:

R²⁰ R²¹ R²² R²³ H benzyl isobutyl H; and methyl isobutyl methylisobutyl.

Exemplary Amino Acid units include, but are not limited to, units offormula (VII) where: R²⁰ is benzyl and R²¹ is —(CH₂)₄NH₂; R²⁰ isopropyland R²¹ is —(CH₂)₄NH₂; R²⁰ isopropyl and R²¹ is —(CH₂)₃NHCONH₂. Anotherexemplary Amino Acid unit is a unit of formula (VIII) wherein R²⁰ isbenzyl, R²¹ is benzyl, and R²² is —(CH₂)₄NH₂.

Useful —W_(w)— units can be designed and optimized in their selectivityfor enzymatic cleavage by a particular enzymes, for example, atumor-associated protease. In one embodiment, a —W_(w)— unit is thatwhose cleavage is catalyzed by cathepsin B, C and D, or a plasminprotease.

In one embodiment, —W_(w)— is a dipeptide, tripeptide, tetrapeptide orpentapeptide.

When R¹⁹, R²⁰, R²¹, R²² or R²³ is other than hydrogen, the carbon atomto which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached is chiral.

Each carbon atom to which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached isindependently in the (S) or (R) configuration.

In one aspect of the Amino Acid unit, the Amino Acid unit isvaline-citrulline. In another aspect, the Amino Acid unit isphenylalanine-lysine (i.e. fk). In yet another aspect of the Amino Acidunit, the Amino Acid unit is N-methylvaline-citrulline. In yet anotheraspect, the Amino Acid unit is 5-aminovaleric acid, homo phenylalaninelysine, tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine,isonepecotic acid lysine, beta-alanine lysine, glycine serine valineglutamine and isonepecotic acid.

In certain embodiments, the Amino Acid unit can comprise natural aminoacids. In other embodiments, the Amino Acid unit can comprisenon-natural amino acids.

The Spacer Unit

The Spacer unit (—Y—), when present, links an Amino Acid unit to theDrug moiety when an Amino Acid unit is present. Alternately, the Spacerunit links the Stretcher unit to the Drug moiety when the Amino Acidunit is absent. The Spacer unit also links the Drug moiety to the Ligandunit when both the Amino Acid unit and Stretcher unit are absent.

Spacer units are of two general types: self-immolative and nonself-immolative. A non self-immolative Spacer unit is one in which partor all of the Spacer unit remains bound to the Drug moiety aftercleavage, particularly enzymatic, of an Amino Acid unit from theDrug-Linker-Ligand Conjugate or the Drug-Linker Compound. Examples of anon self-immolative Spacer unit include, but are not limited to a(glycine-glycine) Spacer unit and a glycine Spacer unit (both depictedin FIG. 20) (infra). When an Exemplary Compound containing aglycine-glycine Spacer unit or a glycine Spacer unit undergoes enzymaticcleavage via a tumor-cell associated-protease, a cancer-cell-associatedprotease or a lymphocyte-associated protease, a glycine-glycine-Drugmoiety or a glycine-Drug moiety is cleaved from L-A_(a)-Ww—. In oneembodiment, an independent hydrolysis reaction takes place within thetarget cell, cleaving the glycine—Drug moiety bond and liberating theDrug.

In another embodiment, —Y_(y)— is a p-aminobenzyl alcohol (PAB) unit(see FIGS. 21 and 22) whose phenylene portion is substituted with Q_(m)wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4.

In one embodiment, a non self-immolative Spacer unit (—Y—) is -Gly-Gly-.

In another embodiment, a non self-immolative the Spacer unit (—Y—) is-Gly-.

In one embodiment, a Drug-Linker Compound or a Drug-Linker LigandConjugate is provided in which the Spacer unit is absent (y=0), or apharmaceutically acceptable salt or solvate thereof.

Alternatively, an Exemplary Compound containing a self-immolative Spacerunit can release -D without the need for a separate hydrolysis step. Inthis embodiment, —Y— is a PAB group that is linked to —W_(w)— via theamino nitrogen atom of the PAB group, and connected directly to -D via acarbonate, carbamate or ether group. Without being bound by anyparticular theory or mechanism, FIG. 21 depicts a possible mechanism ofDrug release of a PAB group which is attached directly to -D via acarbamate or carbonate group espoused by Toki et al. (2002) J. Org.Chem. 67:1866-1872.

In FIG. 21 Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or-cyano; m is an integer ranging from 0-4; and p ranges from 1 to about20.

Without being bound by any particular theory or mechanism, FIG. 22depicts a possible mechanism of Drug release of a PAB group which isattached directly to -D via an ether or amine linkage.

-   -   In FIG. 22 Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -        nitro or -cyano; m is an integer ranging from 0-4; and p ranges        from 1 to about 20.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999)Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals.Spacers can be used that undergo cyclization upon amide bond hydrolysis,such as substituted and unsubstituted 4-aminobutyric acid amides(Rodrigues et al., Chemistry Biology, 1995, 2, 223), appropriatelysubstituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm, etal., J. Amer. Chem. Soc., 1972, 94, 5815) and 2-aminophenylpropionicacid amides (Amsberry, et al., J. Org. Chem., 1990, 55, 5867).Elimination of amine-containing drugs that are substituted at thea-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447)are also examples of self-immolative spacer useful in ExemplaryCompounds.

In one embodiment, the Spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS) unit as depicted in FIG. 23, which canbe used to incorporate and release multiple drugs.

-   -   In FIG. 23 Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro        or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and p        ranges raging from 1 to about 20.

In one embodiment, the -D moieties are the same. In yet anotherembodiment, the -D moieties are different.

In one aspect, Spacer units (—Y_(y)—) are represented by Formulas(X)-(XII):

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;and m is an integer ranging from 0-4;

Embodiments of the Formula Ia′ and Ic antibody-drug conjugate compoundsinclude:

wherein w and y are each 0,

The Drug Unit (Moiety)

The drug moiety (D) of the antibody drug conjugates (ADC) are of thedolastatin/auristatin type (U.S. Pat. Nos. 5,635,483; 5,780,588) whichhave been shown to interfere with microtubule dynamics, GTP hydrolysis,and nuclear and cellular division (Woyke et al. (2001) Antimicrob.Agents and Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat.No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob.Agents Chemother. 42:2961-2965)

D is a Drug unit (moiety) having a nitrogen atom that can form a bondwith the Spacer unit when y=1 or 2, with the C-terminal carboxyl groupof an Amino Acid unit when y=0, with the carboxyl group of a Stretcherunit when w and y=0, and with the carboxyl group of a Drug unit when a,w, and y=0. It is to be understood that the terms “drug unit” and “drugmoiety” are synonymous and used interchangeably herein.

In one embodiment, -D is either formula D_(E) or D_(F):

wherein, independently at each location:

-   -   R² is selected from H and C₁-C₈ alkyl;    -   R³ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁴ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   R⁵ is selected from H and methyl;    -   or R⁴ and R⁵ jointly form a carbocyclic ring and have the        formula —(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are        independently selected from H, C₁-C₈ alkyl and C₃-C₈ carbocycle        and n is selected from 2, 3, 4, 5 and 6;    -   R⁶ is selected from H and C₁-C₈ alkyl;    -   R⁷ is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl,        C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈        heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle);    -   each R⁸ is independently selected from H, OH, C₁-C₈ alkyl, C₃-C₈        carbocycle and O—(C₁-C₈ alkyl);    -   R⁹ is selected from H and C₁-C₈ alkyl;    -   R¹⁰ is selected from aryl or C₃-C₈ heterocycle;    -   Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₈ alkyl;    -   R¹¹ is selected from H, C₁-C₂₀ alkyl, aryl, C₃-C₈ heterocycle,        —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂;    -   m is an integer ranging from 1-1000;    -   R¹³ is C₂-C₈ alkyl;    -   R¹⁴ is H or C₁-C₈ alkyl;    -   each occurrence of R¹⁵ is independently H, COOH,        —(CH₂)_(n)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈        alkyl;    -   each occurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or        —(CH₂)_(n)—COOH;    -   R¹⁸ is selected from —C(R⁸)₂—C(R⁸)₂-aryl, —C(R⁸)₂—C(R⁸)₂—(C₃-C₈        heterocycle), and —C(R⁸)₂—C(R⁸)₂—(C₃-C₈ carbocycle); and    -   n is an integer ranging from 0 to 6.

In one embodiment, R³, R⁴ and R⁷ are independently isopropyl orsec-butyl and R⁵ is —H. In an exemplary embodiment, R³ and R⁴ are eachisopropyl, R⁵ is H, and R⁷ is sec-butyl.

In another embodiment, R² and R⁶ are each methyl, and R⁹ is H.

In still another embodiment, each occurrence of R⁸ is —OCH₃.

In an exemplary embodiment, R³ and R⁴ are each isopropyl, R² and R⁶ areeach methyl, R⁵ is H, R⁷ is sec-butyl, each occurrence of R⁸ is —OCH₃,and R⁹ is H.

In one embodiment, Z is —O— or —NH—.

In one embodiment, R¹⁰ is aryl

In an exemplary embodiment, R¹⁰ is -phenyl.

In an exemplary embodiment, when Z is —O—, R¹¹ is H, methyl or t-butyl.

In one embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is —C₁-C₈ alkyl or —(CH₂)_(n)—COOH.

In another embodiment, when Z is —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is—(CH₂)_(n)—SO₃H. Illustrative Drug units (-D) include the drug unitshaving the following structures:

and pharmaceutically acceptable salts or solvates thereof.

In one aspect, hydrophilic groups, such as but not limited totriethylene glycol esters (TEG), as shown above, can be attached to theDrug Unit at R¹¹. Without being bound by theory, the hydrophilic groupsassist in the internalization and non-agglomeration of the Drug Unit.

The Ligand Unit

The Ligand unit (L-) includes within its scope any unit of a Ligand (L)that binds or reactively associates or complexes with a receptor,antigen or other receptive moiety associated with a given target-cellpopulation. A Ligand is a molecule that binds to, complexes with, orreacts with a moiety of a cell population sought to be therapeuticallyor otherwise biologically modified. In one aspect, the Ligand unit actsto deliver the Drug unit to the particular target cell population withwhich the Ligand unit reacts. Such Ligands include, but are not limitedto, large molecular weight proteins such as, for example, full-lengthantibodies, antibody fragments, smaller molecular weight proteins,polypeptide or peptides, lectins, glycoproteins, non-peptides, vitamins,nutrient-transport molecules (such as, but not limited to, transferrin),or any other cell binding molecule or substance.

A Ligand unit can form a bond to a Stretcher unit, an Amino Acid unit, aSpacer Unit, or a Drug Unit. A Ligand unit can form a bond to a Linkerunit via a heteroatom of the Ligand. Heteroatoms that may be present ona Ligand unit include sulfur (in one embodiment, from a sulfhydryl groupof a Ligand), oxygen (in one embodiment, from a carbonyl, carboxyl orhydroxyl group of a Ligand) and nitrogen (in one embodiment, from aprimary or secondary amino group of a Ligand). These heteroatoms can bepresent on the Ligand in the Ligand's natural state, for example anaturally-occurring antibody, or can be introduced into the Ligand viachemical modification.

In one embodiment, a Ligand has a sulfhydryl group and the Ligand bondsto the Linker unit via the sulfhydryl group's sulfur atom.

In yet another aspect, the Ligand has one or more lysine residues thatcan be chemically modified to introduce one or more sulfhydryl groups.The Ligand unit bonds to the Linker unit via the sulfhydryl group'ssulfur atom. The reagents that can be used to modify lysines include,but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and2-Iminothiolane hydrochloride (Traut's Reagent).

In another embodiment, the Ligand can have one or more carbohydrategroups that can be chemically modified to have one or more sulfhydrylgroups. The Ligand unit bonds to the Linker Unit, such as the StretcherUnit, via the sulfhydryl group's sulfur atom.

In yet another embodiment, the Ligand can have one or more carbohydrategroups that can be oxidized to provide an aldehyde (—CHO) group (see,for e.g., Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). Thecorresponding aldehyde can form a bond with a Reactive Site on aStretcher. Reactive sites on a Stretcher that can react with a carbonylgroup on a Ligand include, but are not limited to, hydrazine andhydroxylamine. Other protocols for the modification of proteins for theattachment or association of Drug Units are described in Coligan et al.,Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002),incorporated herein by reference.

Useful non-immunoreactive protein, polypeptide, or peptide Ligandsinclude, but are not limited to, transferrin, epidermal growth factors(“EGF”), bombesin, gastrin, gastrin-releasing peptide, platelet-derivedgrowth factor, IL-2, IL-6, transforming growth factors (“TGF”), such asTGF-α and TGF-β, vaccinia growth factor (“VGF”), insulin andinsulin-like growth factors I and II, lectins and apoprotein from lowdensity lipoprotein.

Useful polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of immunized animals. Various procedureswell known in the art may be used for the production of polyclonalantibodies to an antigen-of-interest. For example, for the production ofpolyclonal antibodies, various host animals can be immunized byinjection with an antigen of interest or derivative thereof, includingbut not limited to rabbits, mice, rats, and guinea pigs. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and including but not limited to Freund's (completeand incomplete) adjuvant, mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants arealso well known in the art.

Useful monoclonal antibodies are homogeneous populations of antibodiesto a particular antigenic determinant (e.g., a cancer cell antigen, aviral antigen, a microbial antigen, a protein, a peptide, acarbohydrate, a chemical, nucleic acid, or fragments thereof). Amonoclonal antibody (mAb) to an antigen-of-interest can be prepared byusing any technique known in the art which provides for the productionof antibody molecules by continuous cell lines in culture. Theseinclude, but are not limited to, the hybridoma technique originallydescribed by Köhler and Milstein (1975, Nature 256, 495-497), the humanB cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique (Cole et al., 1985, MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Suchantibodies may be of any immunoglobulin class including IgG, IgM, IgE,IgA, and IgD and any subclass thereof. The hybridoma producing the mAbsof use in this invention may be cultivated in vitro or in vivo.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric human-mouse (or other species) monoclonalantibodies. Human monoclonal antibodies may be made by any of numeroustechniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad.Sci. USA. 80, 7308-7312; Kozbor et al., 1983, Immunology Today 4, 72-79;and Olsson et al., 1982, Meth. Enzymol. 92, 3-16).

The antibody can also be a bispecific antibody. Methods for makingbispecific antibodies are known in the art. Traditional production offull-length bispecific antibodies is based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Milstein et al., 1983, Nature 305:537-539).Because of the random assortment of immunoglobulin heavy and lightchains, these hybridomas (quadromas) produce a potential mixture of 10different antibody molecules, of which only one has the correctbispecific structure. Similar procedures are disclosed in InternationalPublication No. WO 93/08829, and in Traunecker et al., EMBO J.10:3655-3659 (1991). According to a different approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, C_(H)2,and C_(H)3 regions. It is preferred to have the first heavy-chainconstant region (C_(H)1) containing the site necessary for light chainbinding, present in at least one of the fusions. Nucleic acids withsequences encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In an embodiment of this approach, the bispecific antibodies have ahybrid immunoglobulin heavy chain with a first binding specificity inone arm, and a hybrid immunoglobulin heavy chain-light chain pair(providing a second binding specificity) in the other arm. Thisasymmetric structure facilitates the separation of the desiredbispecific compound from unwanted immunoglobulin chain combinations, asthe presence of an immunoglobulin light chain in only one half of thebispecific molecule provides for a facile way of separation(International Publication No. WO 94/04690) which is incorporated hereinby reference in its entirety.

For further details for generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology, 1986, 121:210; Rodrigueset al., 1993, J. of Immunology 151:6954-6961; Carter et al., 1992,Bio/Technology 10:163-167; Carter et al., 1995, J. of Hematotherapy4:463-470; Merchant et al., 1998, Nature Biotechnology 16:677-681. Usingsuch techniques, bispecific antibodies can be prepared for use in thetreatment or prevention of disease as defined herein.

Bifunctional antibodies are also described, in European PatentPublication No. EPA 0 105 360. As disclosed in this reference, hybrid orbifunctional antibodies can be derived either biologically, i.e., bycell fusion techniques, or chemically, especially with cross-linkingagents or disulfide-bridge forming reagents, and may comprise wholeantibodies or fragments thereof. Methods for obtaining such hybridantibodies are disclosed for example, in International Publication WO83/03679, and European Patent Publication No. EPA 0 217 577, both ofwhich are incorporated herein by reference.

The antibody can be a functionally active fragment, derivative or analogof an antibody that immunospecifically binds to cancer cell antigens,viral antigens, or microbial antigens or other antibodies bound to tumorcells or matrix. In this regard, “functionally active” means that thefragment, derivative or analog is able to elicit anti-anti-idiotypeantibodies that recognize the same antigen that the antibody from whichthe fragment, derivative or analog is derived recognized. Specifically,in an exemplary embodiment the antigenicity of the idiotype of theimmunoglobulin molecule can be enhanced by deletion of framework and CDRsequences that are C-terminal to the CDR sequence that specificallyrecognizes the antigen. To determine which CDR sequences bind theantigen, synthetic peptides containing the CDR sequences can be used inbinding assays with the antigen by any binding assay method known in theart (e.g., the BIA core assay) (See, for e.g., Kabat et al., 1991,Sequences of Proteins of Immunological Interest, Fifth Edition, NationalInstitute of Health, Bethesda, Md.; Kabat E et al., 1980, J. ofImmunology 125(3):961-969).

Other useful antibodies include fragments of antibodies such as, but notlimited to, F(ab′)₂ fragments, which contain the variable region, thelight chain constant region and the CH1 domain of the heavy chain can beproduced by pepsin digestion of the antibody molecule, and Fabfragments, which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Other useful antibodies are heavy chain and lightchain dimers of antibodies, or any minimal fragment thereof such as Fvsor single chain antibodies (SCAs) (e.g., as described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc.Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature334:544-54), or any other molecule with the same specificity as theantibody.

Additionally, recombinant antibodies, such as chimeric and humanizedmonoclonal antibodies, comprising both human and non-human portions,which can be made using standard recombinant DNA techniques, are usefulantibodies. A chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal and humanimmunoglobulin constant regions. (See, e.g., Cabilly et al., U.S. Pat.No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397, which areincorporated herein by reference in their entirety.) Humanizedantibodies are antibody molecules from non-human species having one ormore complementarity determining regions (CDRs) from the non-humanspecies and a framework region from a human immunoglobulin molecule.(See, e.g., Queen, U.S. Pat. No. 5,585,089, which is incorporated hereinby reference in its entirety.) Such chimeric and humanized monoclonalantibodies can be produced by recombinant DNA techniques known in theart, for example using methods described in International PublicationNo. WO 87/02671; European Patent Publication No. 184,187; EuropeanPatent Publication No. 171496; European Patent Publication No. 173494;International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;European Patent Publication No. 12,023; Berter et al., 1988, Science240:1041-1043; Liu et al., 1987, Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al.,1987, Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al., 1987,Cancer. Res. 47:999-1005; Wood et al., 1985, Nature 314:446-449; andShaw et al., 1988, J. Natl. Cancer Inst. 80:1553-1559; Morrison, 1985,Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; U.S. Pat.No. 5,225,539; Jones et al., 1986, Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al., 1988, J. Immunol.141:4053-4060; each of which is incorporated herein by reference in itsentirety.

Completely human antibodies are particularly desirable and can beproduced using transgenic mice that are incapable of expressingendogenous immunoglobulin heavy and light chains genes, but which canexpress human heavy and light chain genes. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained using conventionalhybridoma technology. The human immunoglobulin transgenes harbored bythe transgenic mice rearrange during B cell differentiation, andsubsequently undergo class switching and somatic mutation. Thus, usingsuch a technique, it is possible to produce therapeutically useful IgG,IgA, IgM and IgE antibodies. For an overview of this technology forproducing human antibodies, see Lonberg and Huszar (1995, Int. Rev.Immunol. 13:65-93). For a detailed discussion of this technology forproducing human antibodies and human monoclonal antibodies and protocolsfor producing such antibodies. See, e.g., U.S. Pat. Nos. 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; each of which isincorporated herein by reference in its entirety. Other human antibodiescan be obtained commercially from, for example, Abgenix, Inc. (Freemont,Calif.) and Genpharm (San Jose, Calif.).

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al. (1994) Biotechnology12:899-903). Human antibodies can also be produced using varioustechniques known in the art, including phage display libraries(Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J.Mol. Biol., 222:581 (1991); Quan, M. P. and Carter, P. 2002. The rise ofmonoclonal antibodies as therapeutics. In Anti-IgE and Allergic Disease,Jardieu, P. M. and Fick Jr., R. B, eds., Marcel Dekker, New York, N.Y.,Chapter 20, pp. 427-469).

In other embodiments, the antibody is a fusion protein of an antibody,or a functionally active fragment thereof, for example in which theantibody is fused via a covalent bond (e.g., a peptide bond), at eitherthe N-terminus or the C-terminus to an amino acid sequence of anotherprotein (or portion thereof, preferably at least 10, 20 or 50 amino acidportion of the protein) that is not the antibody. Preferably, theantibody or fragment thereof is covalently linked to the other proteinat the N-terminus of the constant domain.

Antibodies include analogs and derivatives that are either modified,i.e., by the covalent attachment of any type of molecule as long as suchcovalent attachment permits the antibody to retain its antigen bindingimmunospecificity. For example, but not by way of limitation, thederivatives and analogs of the antibodies include those that have beenfurther modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular antibody unit orother protein, etc. Any of numerous chemical modifications can becarried out by known techniques, including, but not limited to specificchemical cleavage, acetylation, formylation, metabolic synthesis in thepresence of tunicamycin, etc. Additionally, the analog or derivative cancontain one or more unnatural amino acids.

The antibodies include antibodies having modifications (e.g.,substitutions, deletions or additions) in amino acid residues thatinteract with Fc receptors. In particular, antibodies include antibodieshaving modifications in amino acid residues identified as involved inthe interaction between the anti-Fc domain and the FcRn receptor (see,e.g., International Publication No. WO 97/34631, which is incorporatedherein by reference in its entirety). Antibodies immunospecific for acancer cell antigen can be obtained commercially, for example, fromGenentech (San Francisco, Calif.) or produced by any method known to oneof skill in the art such as, e.g., chemical synthesis or recombinantexpression techniques. The nucleotide sequence encoding antibodiesimmunospecific for a cancer cell antigen can be obtained, e.g., from theGenBank database or a database like it, the literature publications, orby routine cloning and sequencing.

In a specific embodiment, known antibodies for the treatment orprevention of cancer can be used. Antibodies immunospecific for a cancercell antigen can be obtained commercially or produced by any methodknown to one of skill in the art such as, e.g., recombinant expressiontechniques. The nucleotide sequence encoding antibodies immunospecificfor a cancer cell antigen can be obtained, e.g., from the GenBankdatabase or a database like it, the literature publications, or byroutine cloning and sequencing. Examples of antibodies available for thetreatment of cancer include, but are not limited to, humanized anti-HER2monoclonal antibody, HERCEPTIN® (trastuzumab; Genentech) for thetreatment of patients with metastatic breast cancer; RITUXAN®(rituximab; Genentech) which is a chimeric anti-CD20 monoclonal antibodyfor the treatment of patients with non-Hodgkin's lymphoma; OvaRex(AltaRex Corporation, MA) which is a murine antibody for the treatmentof ovarian cancer; Panorex (Glaxo Wellcome, NC) which is a murineIgG_(2a) antibody for the treatment of colorectal cancer; CetuximabErbitux (Imclone Systems Inc., NY) which is an anti-EGFR IgG chimericantibody for the treatment of epidermal growth factor positive cancers,such as head and neck cancer; Vitaxin (MedImmune, Inc., MD) which is ahumanized antibody for the treatment of sarcoma; Campath I/H (Leukosite,MA) which is a humanized IgG₁ antibody for the treatment of chroniclymphocytic leukemia (CLL); Smart MI95 (Protein Design Labs, Inc., CA)which is a humanized anti-CD33 IgG antibody for the treatment of acutemyeloid leukemia (AML); LymphoCide (Immunomedics, Inc., NJ) which is ahumanized anti-CD22 IgG antibody for the treatment of non-Hodgkin'slymphoma; Smart ID10 (Protein Design Labs, Inc., CA) which is ahumanized anti-HLA-DR antibody for the treatment of non-Hodgkin'slymphoma; Oncolym (Techniclone, Inc., CA) which is a radiolabeled murineanti-HLA-Dr10 antibody for the treatment of non-Hodgkin's lymphoma;Allomune (BioTransplant, CA) which is a humanized anti-CD2 mAb for thetreatment of Hodgkin's Disease or non-Hodgkin's lymphoma; Avastin(Genentech, Inc., CA) which is an anti-VEGF humanized antibody for thetreatment of lung and colorectal cancers; Epratuzamab (Immunomedics,Inc., NJ and Amgen, CA) which is an anti-CD22 antibody for the treatmentof non-Hodgkin's lymphoma; and CEAcide (Immunomedics, NJ) which is ahumanized anti-CEA antibody for the treatment of colorectal cancer.

Other antibodies useful in the treatment of cancer include, but are notlimited to, antibodies against the following antigens: CA125 (ovarian),CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), Lewis Y(carcinomas), Lewis X (carcinomas), alpha fetoprotein (carcinomas), CA242 (colorectal), placental alkaline phosphatase (carcinomas), prostatespecific antigen (prostate), prostatic acid phosphatase (prostate),epidermal growth factor (carcinomas), MAGE-1 (carcinomas), MAGE-2(carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), anti-transferrinreceptor (carcinomas), p97 (melanoma), MUC1-KLH (breast cancer), CEA(colorectal), gp100 (melanoma), MART1 (melanoma), PSA (prostate), IL-2receptor (T-cell leukemia and lymphomas), CD20 (non-Hodgkin's lymphoma),CD52 (leukemia), CD33 (leukemia), CD22 (lymphoma), human chorionicgonadotropin (carcinoma), CD38 (multiple myeloma), CD40 (lymphoma),mucin (carcinomas), P21 (carcinomas), MPG (melanoma), and Neu oncogeneproduct (carcinomas).

Some specific, useful antibodies include, but are not limited to, BR96mAb (Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J.,Hofstead, S. J., Casazza, A. M., Firestone, R. A., Hellström, I.,Hellström, K. E., “Cure of Xenografted Human Carcinomas byBR96-Doxorubicin Immunoconjugates” Science 1993, 261, 212-215), BR64(Trail, P A, Willner, D, Knipe, J., Henderson, A. J., Lasch, S. J.,Zoeckler, M. E., Trailsmith, M. D., Doyle, T. W., King, H. D., Casazza,A. M., Braslawsky, G. R., Brown, J. P., Hofstead, S. J., (Greenfield, R.S., Firestone, R. A., Mosure, K., Kadow, D. F., Yang, M. B., Hellstrom,K. E., and Hellstrom, I. “Effect of Linker Variation on the Stability,Potency, and Efficacy of Carcinoma-reactive BR64-DoxorubicinImmunoconjugates” Cancer Research 1997, 57, 100-105, mAbs against theCD40 antigen, such as S2C6 mAb (Francisco, J. A., Donaldson, K. L.,Chace, D., Siegall, C. B., and Wahl, A. F. “Agonistic properties and invivo antitumor activity of the anti-CD-40 antibody, SGN-14” Cancer Res.2000, 60, 3225-3231), mAbs against the CD70 antigen, such as 1F6 mAb and2F2 mAb, and mAbs against the CD30 antigen, such as AC10 (Bowen, M. A.,Olsen, K. J., Cheng, L., Avila, D., and Podack, E. R. “Functionaleffects of CD30 on a large granular lymphoma cell line YT” J. Immunol.,151, 5896-5906, 1993: Wahl et al., 2002 Cancer Res. 62(13):3736-42).Many other internalizing antibodies that bind to tumor associatedantigens can be used and have been reviewed (Franke, A. E., Sievers, E.L., and Scheinberg, D. A., “Cell surface receptor-targeted therapy ofacute myeloid leukemia: a review” Cancer Biother Radiopharm. 2000, 15,459-76; Murray, J. L., “Monoclonal antibody treatment of solid tumors: acoming of age” Semin Oncol. 2000, 27, 64-70; Breitling, F., and Dubel,S., Recombinant Antibodies, John Wiley, and Sons, New York, 1998).

In certain embodiments, the antibody is not Trastuzumab (full length,humanized anti-HER2 (MW 145167)), HerceptinF(ab′)₂ (derived fromanti-HER2 enzymatically (MW 100000)), 4D5 (full-length, murine antiHER2,from hybridoma), rhu4D5 (transiently expressed, full-length humanizedantibody), rhuFab4D5 (recombinant humanized Fab (MW 47738)), 4D5Fc8(full-length, murine antiHER2, with mutated FcRn binding domain), or Hg(“Hingeless” full-length humanized 4D5, with heavy chain hinge cysteinesmutated to serines. Expressed in E. coli (therefore non-glycosylated)).

In another specific embodiment, known antibodies for the treatment orprevention of an autoimmune disease are used in accordance with thecompositions and methods of the invention. Antibodies immunospecific foran antigen of a cell that is responsible for producing autoimmuneantibodies can be obtained from any organization (e.g., a universityscientist or a company) or produced by any method known to one of skillin the art such as, e.g., chemical synthesis or recombinant expressiontechniques. In another embodiment, useful antibodies are immunospecificfor the treatment of autoimmune diseases include, but are not limitedto, Anti-Nuclear Antibody; Anti-ds DNA; Anti-ss DNA, Anti-CardiolipinAntibody IgM, IgG; Anti-Phospholipid Antibody IgM, IgG; Anti-SMAntibody; Anti-Mitochondrial Antibody; Thyroid Antibody; MicrosomalAntibody; Thyroglobulin Antibody; Anti-SCL-70; Anti-Jo; Anti-U₁RNP;Anti-La/SSB; Anti SSA; Anti-SSB; Anti-Perital Cells Antibody;Anti-Histones; Anti-RNP; C-ANCA; P-ANCA; Anti centromere;Anti-Fibrillarin, and Anti-GBM Antibody.

In certain embodiments, useful antibodies can bind to both a receptor ora receptor complex expressed on an activated lymphocyte. The receptor orreceptor complex can comprise an immunoglobulin gene superfamily member,a TNF receptor superfamily member, an integrin, a cytokine receptor, achemokine receptor, a major histocompatibility protein, a lectin, or acomplement control protein. Non-limiting examples of suitableimmunoglobulin superfamily members are CD2, CD3, CD4, CD8, CD19, CD22,CD28, CD79, CD90, CD152/CTLA-4, PD-1, and ICOS. Non-limiting examples ofsuitable TNF receptor superfamily members are CD27, CD40, CD95/Fas,CD134/OX40, CD137/4-1BB, TNF-R1, TNFR-2, RANK, TACI, BCMA,osteoprotegerin, Apo2/TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3.Non-limiting examples of suitable integrins are CD11a, CD11b, CD11c,CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD103, andCD104. Non-limiting examples of suitable lectins are C-type, S-type, andI-type lectin.

In one embodiment, the Ligand binds to an activated lymphocyte that isassociated with an autoimmune disease.

In another specific embodiment, useful Ligands immunospecific for aviral or a microbial antigen are monoclonal antibodies. The antibodiesmay be chimeric, humanized or human monoclonal antibodies. As usedherein, the term “viral antigen” includes, but is not limited to, anyviral peptide, polypeptide protein (e.g., HIV gp120, HIV nef, RSV Fglycoprotein, influenza virus neuraminidase, influenza virushemagglutinin, HTLV tax, herpes simplex virus glycoprotein (e.g., gB,gC, gD, and gE) and hepatitis B surface antigen) that is capable ofeliciting an immune response. As used herein, the term “microbialantigen” includes, but is not limited to, any microbial peptide,polypeptide, protein, saccharide, polysaccharide, or lipid molecule(e.g., a bacterial, fungi, pathogenic protozoa, or yeast polypeptideincluding, e.g., LPS and capsular polysaccharide 5/8) that is capable ofeliciting an immune response. Antibodies immunospecific for a viral ormicrobial antigen can be obtained commercially, for example, from BDBiosciences (San Francisco, Calif.), Chem icon International, Inc.(Temecula, Calif.), or Vector Laboratories, Inc. (Burlingame, Calif.) orproduced by any method known to one of skill in the art such as, e.g.,chemical synthesis or recombinant expression techniques. The nucleotidesequence encoding antibodies that are immunospecific for a viral ormicrobial antigen can be obtained, e.g., from the GenBank database or adatabase like it, literature publications, or by routine cloning andsequencing. In a specific embodiment, useful Ligands are those that areuseful for the treatment or prevention of viral or microbial infectionin accordance with the methods disclosed herein. Examples of antibodiesavailable useful for the treatment of viral infection or microbialinfection include, but are not limited to, SYNAGIS (MedImmune, Inc., MD)which is a humanized anti-respiratory syncytial virus (RSV) monoclonalantibody useful for the treatment of patients with RSV infection; PRO542(Progenies) which is a CD4 fusion antibody useful for the treatment ofHIV infection; OSTAVIR (Protein Design Labs, Inc., CA) which is a humanantibody useful for the treatment of hepatitis B virus; PROTOVIR(Protein Design Labs, Inc., CA) which is a humanized IgG₁ antibodyuseful for the treatment of cytomegalovirus (CMV); and anti-LPSantibodies.

Other antibodies useful in the treatment of infectious diseases include,but are not limited to, antibodies against the antigens from pathogenicstrains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae,Neisseria gonorrheae, Neisseria men ingitidis, Corynebacteriumdiphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridiumtetani, Hemophilus influenzae, Klebsiella pneumoniae, Klebsiellaozaenas, Klebsiella rhinoscleromotis, Staphylococc aureus, Vibriocolerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter(Vibrio) fetus, Aeromonas hydrophila, Bacillus cereus, Edwardsiellatarda, Yersinia enterocolitica, Yersinia pestis, Yersiniapseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigellasonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue,Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi,Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Pneumocystiscarinii, Francisella tularensis, Brucella abortus, Brucella suis,Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsiatsutsugumushi, Chlamydia spp.); pathogenic fungi (Coccidioides immitis,Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis,Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entomoebahistolytica, Toxoplasma gondii, Trichomonas tenas, Trichomonas hominis,Trichomonas vaginalis, Tryoanosoma gambiense, Trypanosoma rhodesiense,Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmaniabraziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodiumfalciparum, Plasmodium malaria); or Helminiths (Enterobius vermicularis,Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis,Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni,Schistosoma haematobium, and hookworms).

Other antibodies useful in this invention for treatment of viral diseaseinclude, but are not limited to, antibodies against antigens ofpathogenic viruses, including as examples and not by limitation:Poxyiridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae,Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenzaviruses, mumps, measles, respiratory syncytial virus, rubella,Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis Bvirus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitisvirus, Rhinoviridae, Coronaviridae, Rotoviridae, and HumanImmunodeficiency Virus.

In attempts to discover effective cellular targets for cancer diagnosisand therapy, researchers have sought to identify transmembrane orotherwise tumor-associated polypeptides that are specifically expressedon the surface of one or more particular type(s) of cancer cell ascompared to on one or more normal non-cancerous cell(s). Often, suchtumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies.

Antibodies which comprise Ab in Formula Ic antibody drug conjugates(ADC) and which may be useful in the treatment of cancer include, butare not limited to, antibodies against tumor-associated antigens (TAA).Such tumor-associated antigens are known in the art, and can preparedfor use in generating antibodies using methods and information which arewell known in the art. Examples of TAA include (1)-(35), but are notlimited to TAA (1)-(35) listed below. For convenience, informationrelating to these antigens, all of which are known in the art, is listedbelow and includes names, alternative names, Genbank accession numbersand primary reference(s). Tumor-associated antigens targeted byantibodies include all amino acid sequence variants and isoformspossessing at least about 70%, 80%, 85%, 90%, or 95% sequence identityrelative to the sequences identified in the corresponding sequenceslisted (SEQ ID NOS: 1-35) or the sequences identified in the citedreferences. In some embodiments, TAA having amino acid sequence variantsexhibit substantially the same biological properties or characteristicsas a TAA having the sequence found in the corresponding sequences listed(SEQ ID NOS: 1-35). For example, a TAA having a variant sequencegenerally is able to bind specifically to an antibody that bindsspecifically to the TAA with the corresponding sequence listed. Thesequences and disclosure specifically recited herein are expresslyincorporated by reference.

TUMOR-ASSOClATED ANTIGENS (1)-(35):

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM_(—)001203, ten Dijke, P., et al. Science 264(5155):101-104 (1994), Oncogene 14(11):1377-1382 (1997)); WO2004063362(claim 2); WO2003042661 (claim 12); US2003134790-A1 (Page 38-39);WO2002102235 (claim 13; Page 296); WO2003055443 (Page 91-92);WO200299122 (Example 2; Page 528-530); WO2003029421 (claim 6);WO2003024392 (claim 2; FIG. 112); WO200298358 (claim 1; Page 183);WO200254940 (Page 100-101); WO200259377(Page 349-350); WO200230268(claim 27; Page 376); WO200148204 (Example; FIG. 4)NP_(—)001194 bone morphogenetic protein receptor, typeIB/pid=NP_(—)001194.1-Cross-references: MIM:603248; NP_(—)001194.1;NM_(—)001203_(—)1

502 aa (SEQ ID NO: 1)MLLRSAGKLNVGTKKEDGESTAPTPRPKVLRCKCHHHCPEDSVNNICSTDGYCFTMIEEDDSGLPVVTSGCLGLEGSDFQCRDTPIPHQRRSIECCTERNECNKDLHPTLPPLKNRDFVDGPIHHRALLISVTVCSLLLVLIILFCYFRYKRQETRPRYSIGLEQDETYIPPGESLRDLIEQSQSSGSGSGLPLLVQRTIAKQIQMVKQIGKGRYGEVWMGKWRGEKVAVKVFFTTEEASWFRETEIYQTVLMRHENILGFIAADIKGTGSWTQLYLITDYHENGSLYDYLKSTTLDAKSMLKLAYSSVSGLCHLHTEIFSTQGKPAIAHRDLKSKNILVKKNGTCCIADLGLAVKFISDTNEVDIPPNTRVGTKRYMPPEVLDESLNRNHFQSYIMADMYSFGLILWEVARRCVSGGIVEEYQLPYHDLVPSDPSYEDMREIVCIKKLRPSFPNRWSSDECLRQMGKLMTECWAHNPASRLTALRVKKTLAKMSESQDIKL(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_(—)003486); Biochem.Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291(1998), Gaugitsch, H. W., et al. (1992) J. Biol. Chem. 267(16):11267-11273); WO2004048938 (Example 2); WO2004032842 (Example IV);WO2003042661 (claim 12); WO2003016475 (claim 1); WO200278524 (Example2); WO200299074 (claim 19; Page 127-129); WO200286443 (claim 27; Pages222, 393); WO2003003906 (claim 10; Page 293); WO200264798 (claim 33;Page 93-95); WO200014228 (claim 5; Page 133-136); US2003224454 (FIG. 3);WO2003025138 (claim 12; Page 150);NP_(—)003477 solute carrier family 7 (cationic amino acid transporter,y+ system), member 5/pid=NP_(—)003477.3—Homo sapiens

Cross-references: MIM:600182; NP_(—)003477.3; NM_(—)015923;NM_(—)003486_(—)1

507 aa (SEQ ID NO: 2)MAGAGPKRRALAAPAAEEKEEAREKMLAAKSADGSAPAGEGEGVTLQRNITLLNGVAIIVGTIIGSGIFVTPTGVLKEAGSPGLALVVWAACGVFSIVGALCYAELGTTISKSGGDYAYMLEVYGSLPAFLKLWIELLIIRPSSQYIVALVFATYLLKPLFPTCPVPEEAAKLVACLCVLLLTAVNCYSVKAATRVQDAFAAAKLLALALIILLGFVQIGKGVVSNLDPNFSFEGTKLDVGNIVLALYSGLFAYGGWNYLNFVTEEMINPYRNLPLAIIISLPIVTLVYVLTNLAYFTTLSTEQMLSSEAVAVDFGNYHLGVMSWIIPVFVGLSCFGSVNGSLFTSSRLFFVGSREGHLPSILSMIHPQLLTPVPSLVFTCVMTLLYAFSKDIFSVINFFSFFNWLCVALAIIGMIWLRHRKPELERPIKVNLALPVFFILACLFLIAVSFWKTPVECGIGFTIILSGLPVYFFGVWWKNKPKWLLQGIFSTTVLCQKLMQVVPQET(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbankaccession no. NM_(—)012449Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al. (1999)Proc. Natl. Acad. Sci. USA. 96 (25):14523-14528); WO2004065577 (claim6); WO2004027049 (FIG. 1L); EP1394274 (Example 11); WO2004016225 (claim2); WO2003042661 (claim 12); US2003157089 (Example 5); US2003185830(Example 5); US2003064397 (FIG. 2); WO200289747 (Example 5; Page618-619); WO2003022995 (Example 9; FIG. 13A, Example 53; Page 173,Example 2; FIG. 2A);NP_(—)036581 six transmembrane epithelial antigen of the prostate

Cross-references: MIM:604415; NP_(—)036581.1; NM_(—)012449_(—)1

339 aa (SEQ ID NO 3)MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRKKILKIRHGWEDVTKINKTEICSQL(4) 0772P (CA125, MUC16, Genbank accession no. AF361486J. Biol. Chem. 276 (29):27371-27375 (2001)); WO2004045553 (claim 14);WO200292836 (claim 6; FIG. 12); WO200283866 (claim 15; Page 116-121);US2003124140 (Example 16); US2003091580 (claim 6); WO200206317 (claim 6;Page 400-408);

Cross-references: GI:34501467; AAK74120.3; AF361486_(—1)

6995 aa (SEQ ID NO: 4)PVTSLLTPGLVITTDRMGISREPGTSSTSNLSSTSHERLTTLEDTVDTEAMQPSTHTAVTNVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSLTSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPDISTEAITRLSTSPIMTESAESAITIETGSPGATSEGTLTLDTSTTTFWSGTHSTASPGFSHSEMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFFSTLPESISSSPHPVTALLTLGPVKTTDMLRTSSEPETSSPPNLSSTSAEILATSEVTKDREKIHPSSNTPVVNVGTVIYKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLREISTSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRISTPLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSRGPEDVSWPSRPSVEKTSPPSSLVSLSAVTSPSPLYSTPSESSHSSPLRVTSLFTPVMMKTTDMLDTSLEPVTTSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYSSYPGLPEPSKVTSPVVTSSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETSTSQEIHSATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISTEVITRLSTSPIKTESTEMTITTQTGSPGATSRGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGEVPWLSHPSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSEPETSSPPNLSSTSAEILATTEVTTDTEKLEMTNVVTSGYTHESPSSVLADSVTTKATSSMGITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSVPTGATTEVSRTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRKESTDMAITPKTGPSGATSQGTFTLDSSSTASWPGTHSATTQRFPRSVVTTPMSRGPEDVSWPSPLSVEKNSPPSSLVSSSSVTSPSPLYSTPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNITSDESLAASKATTETEAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPVVTSSSIRDNMVSTTMPGSSGITRIEIESMSSLTPGLRETRTSQDITSSTETSTVLYKMPSGATPEVSRTEVMPSSRTSIPGPAQSTMSLDISDEVVTRLSTSPIMTESAEITITTQTGYSLATSQVTLPLGTSMTFLSGTHSTMSQGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLPLTTSLSPVSSTLLDSSPSSPLPVTSLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVEIPATSEIMTDTEKIHPSSNTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVEDTTVFTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLFGVPTSATTEVSMTEIMSSNRTHIPDSDQSTMSPDIITEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLTLATTTAPLARTHSTVPPRFLHSEMTTLMSRSPENPSWKSSPFVEKTSSSSSLLSLPVTTSPSVSSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEILAASEVTTDTEKIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANFETTGFEAEPFSHLTSGLRKTNMSLDTSSVTPTNTPSSPGSTHLLQSSKTDFTSSAKTSSPDWPPASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAETISTGTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPFSSSTFTTTDSSTIPALHEITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSSPILDTRMTESVELGTVTSAYQVPSLSTRLTRTDGIMEHITKIPNEAAHRGTIRPVKGPQTSTSPASPKGLHTGGTKRMETTTTALKTTTTALKTTSRATLTTSVYTPTLGTLTPLNASMQMASTIPTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVFNRESETTASLVSRSGAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHSELDTVSSTATSHGADVSSAIPTNISPSELDALTPLVTISGTDTSTTFPTLTKSPHETETRTTWLTHPAETSSTIPRTIPNFSHHESDATPSIATSPGAETSSAIPIMTVSPGAEDLVTSQVTSSGTDRNMTIPTLTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSLAAKTSTTNRALTNSPGEPATTVSLVTHSAQTSPTVPWTTSIFFHSKSDTTPSMTTSHGAESSSAVPTPTVSTEVPGVVTPLVTSSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGVVTSLVTSSRAVTSTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTVLPEVPGMVTSLVASSRAVTSTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGVVTSLVTSSSGVNSTSIPTLILSPGELETTPSMATSHGAEASSAVPTPTVSPGVSGVVTPLVTSSRAVTSTTIPILTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTFLVTSSRAVTSTTIPTLTISSDEPETTTSLVTHSEAKMISAIPTLGVSPTVQGLVTSLVTSSGSETSAFSNLTVASSQPETIDSWVAHPGTEASSVVPTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTMPSTVTSPEAESSSAISTTISPGIPGVLTSLVTSSGRDISATFPTVPESPHESEATASWVTHPAVTSTTVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPTITVSPDVPDMVTSQVTSSGTDTSITIPTLTLSSGEPETTTSFITYSETHTSSAIPTLPVSPDASKMLTSLVISSGTDSTTTFPTLTETPYEPETTAIQLIHPAETNTMVPRTTPKFSHSKSDTTLPVAITSPGPEASSAVSTTTISPDMSDLVTSLVPSSGTDTSTTFPTLSETPYEPETTATWLTHPAETSTTVSGTIPNFSHRGSDTAPSMVTSPGVDTRSGVPTTTIPPSIPGVVTSQVTSSATDTSTAIPTLTPSPGEPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSSPDATPVMATSPRTEASSAVLTTISPGAPEMVTSQITSSGAATSTTVPTLTHSPGMPETTALLSTHPRTETSKTFPASTVFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVDLSPTASPGVSAKTAPLSTHPGTETSTMIPTSTLSLGLLETTGLLATSSSAETSTSTLTLTVSPAVSGLSSASITTDKPQTVTSWNTETSPSVTSVGPPEFSRTVTGTTMTLIPSEMPTPPKTSHGEGVSPTTILRTTMVEATNLATTGSSPTVAKTTTTFNTLAGSLFTPLTTPGMSTLASESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTSSIPSSTAATVPFMVPFTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSATAVDAICTHRPDPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRSSMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLMPFTLNFTITNLQYEEDMRRTGSRKFNTMESVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLTNDIEELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSSLSSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRTSVPTTSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITNLKYEEDMHRPGSRKFNTTERVLQTLVGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTSATAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLLGPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPYTLDRNSLYVNGFTHRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTTAVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSPGLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTPWTSTVDLGTSGTPSPVPSPTTAGPLLVPFTLNFTITNLQYEEDMHRPGSRKFNATERVLQGLLSPIFKNSSVGPLYSGCRLTSLRPEKDGAATGMDAVCLYHPNPKRPGLDREQLYWELSQLTHNITELGPYSLDRDSLYVNGFTHQNSVPTTSTPGTSTVYWATTGTPSSFPGHTEPGPLLIPFTFNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKQEAATGVDTICTHRVDPIGPGLDRERLYWELSQLTNSITELGPYTLDRDSLYVNGFNPWSSVPTTSTPGTSTVHLATSGTPSSLPGHTAPVPLLIPFTLNFTITNLHYEENMQHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKHGAATGVDAICTLRLDPTGPGLDRERLYWELSQLTNSVTELGPYTLDRDSLYVNGFTHRSSVPTTSIPGTSAVHLETSGTPASLPGHTAPGPLLVPFTLNFTITNLQYEEDMRHPGSRKFNTTERVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKRGAATGVDTICTHRLDPLNPGLDREQLYWELSKLTRGIIELGPYLLDRGSLYVNGFTHRNFVPITSTPGTSTVHLGTSETPSSLPRPIVPGPLLVPFTLNFTITNLQYEEAMRHPGSRKFNTTERVLQGLLRPLFKNTSIGPLYSSCRLTLLRPEKDKAATRVDAICTHHPDPQSPGLNREQLYWELSQLTHGITELGPYTLDRDSLYVDGFTHWSPIPTTSTPGTSIVNLGTSGIPPSLPETTATGPLLVPFTLNFTITNLQYEENMGHPGSRKFNITESVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFTIINLQYEEDMHRPGSRKFNTTERVLQGLLMPLFKNTSVSSLYSGCRLTLLRPEKDGAATRVDAVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPTTASPLLVLFTINFTITNLRYEENMHHPGSRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASPLLVLFTLNFTITNLRYEENMQHPGSRKFNTTERVLQGLLRSLFKSTSVGPLYSGCRLTLLRPEKDGTATGVDAICTHHPDPKSPRLDREQLYWELSQLTHNITELGPYALDNDSLFVNGFTHRSSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLILFTLNFTITNLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGCRLTLLRPEKDGEATGVDAICTHRPDPTGPGLDREQLYLELSQLTHSITELGPYTLDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTINNLRYMADMGQPGSLKFNITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRVDLLCTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGPDEPPTTPKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRPLFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHGVTQLGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTSSEYITLLRDIQDKVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTVKALFSSNLDPSLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGLITCLICGVLVTTRRRKKEGEYNVQQQCPGYYQSHLDLEDLQ(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin,Genbank accession no. NM_(—)005823Yamaguchi, N., et al. Biol. Chem. 269 (2), 805-808 (1994), Proc. Natl.Acad. Sci. USA. 96 (20):11531-11536 (1999), Proc. Natl. Acad. Sci. USA.93 (1):136-140 (1996), J. Biol. Chem. 270 (37):21984-21990 (1995));WO2003101283 (claim 14); (WO2002102235 (claim 13; Page 287-288);WO2002101075 (claim 4; Page 308-309); WO200271928 (Page 320-321);WO9410312 (Page 52-57);

Cross-references: MIM:601051; NP_(—)005814.2; NM_(—)005823_(—1)

622 aa (SEQ ID NO: 5)MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter 3b,Genbank accession no. NM_(—)006424,J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284(1999), Feild, J. A., et al. (1999) Biochem. Biophys. Res. Commun. 258(3):578-582); WO2004022778 (claim 2); EP1394274 (Example 11);WO2002102235 (claim 13; Page 326); EP875569 (claim 1; Page 17-19);WO200157188 (claim 20; Page 329); WO2004032842 (Example IV); WO200175177(claim 24; Page 139-140);

Cross-references: MIM:604217; NP_(—)006415.1; NM_(—)006424_(—)1

690 aa (SEQ ID NO: 6)MAPWPELGDAQPNPDKYLEGAAGQQPTAPDKSKETNKTDNTEAPVTKIELLPSYSTATLIDEPTEVDDPWNLPTLQDSGIKWSERDTKGKILCFFQGIGRLILLLGFLYFFVCSLDILSSAFQLVGGKMAGQFFSNSSIMSNPLLGLVIGVLVTVLVQSSSTSTSIVVSMVSSSLLTVRAAIPIIMGANIGTSITNTIVALMQVGDRSEFRRAFAGATVHDFFNWLSVLVLLPVEVATHYLEIITQLIVESFHFKNGEDAPDLLKVITKPFTKLIVQLDKKVISQIAMNDEKAKNKSLVKIWCKTFTNKTQINVTVPSTANCTSPSLCWTDGIQNWTMKNVTYKENIAKCQHIFVNFHLPDLAVGTILLILSLLVLCGCLIMIVKILGSVLKGQVATVIKKTINTDFPFPFAWLTGYLAILVGAGMTFIVQSSSVFTSALTPLIGIGVITIERAYPLTLGSNIGTTTTAILAALASPGNALRSSLQIALCHFFFNISGILLWYPIPFTRLPIRMAKGLGNISAKYRWFAVFYLIIFFFLIPLTVFGLSLAGWRVLVGVGVPVVFIIILVLCLRLLQSRCPRVLPKKLQNWNFLPLWMRSLKPWDAVVSKFTGCFQMRCCYCCRVCCRACCLLCGCPKCCRCSKCCEDLEEAQEGQDVPVKAPETFDNITISREAQGEVPASDSKTECTAL(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMASB, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B, Genbank accession no. AB040878, Nagase T., et al.(2000) DNA Res. 7 (2):143-150); WO2004000997 (claim 1); WO2003003984(claim 1); WO200206339 (claim 1; Page 50); WO200188133 (claim 1; Page41-43, 48-58); WO2003054152 (claim 20); WO2003101400 (claim 11);Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC:10737;

1093 aa (SEQ ID NO: 7)MVLAGPLAVSLLLPSLTLLVSHLSSSQDVSSEPSSEQQLCALSKHPTVAFEDLQPWVSNFTYPGARDFSQLALDPSGNQLIVGARNYLFRLSLANVSLLQATEWASSEDTRRSCQSKGKTEEECQNYVRVLIVAGRKVFMCGTNAFSPMCTSRQVGNLSRTTEKINGVARCPYDPRHNSTAVISSQGELYAATVIDFSGRDPAIYRSLGSGPPLRTAQYNSKWLNEPNFVAAYDIGLFAYFFLRENAVEHDCGRTVYSRVARVCKNDVGGRFLLEDTWTTFMKARLNCSRPGEVPFYYNELQSAFHLPEQDLIYGVFTTNVNSIAASAVCAFNLSAISQAFNGPFRYQENPRAAWLPIANPIPNFQCGTLPETGPNENLTERSLQDAQRLFLMSEAVQPVTPEPCVTQDSVRFSHLVVDLVQAKDTLYHVLYIGTESGTILKALSTASRSLHGCYLEELHVLPPGRREPLRSLRILHSARALFVGLRDGVLRVPLERCAAYRSQGACLGARDPYCGWDGKQQRCSTLEDSSNMSLWTQNITACPVRNVTRDGGFGPWSPWQPCEHLDGDNSGSCLCRARSCDSPRPRCGGLDCLGPAIHIANCSRNGAWTPWSSWALCSTSCGIGFQVRQRSCSNPAPRHGGRICVGKSREERFCNENTPCPVPIFWASWGSWSKCSSNCGGGMQSRRRACENGNSCLGCGVEFKTCNPEGCPEVRRNTPWTPWLPVNVTQGGARQEQRFRFTCRAPLADPHGLQFGRRRTETRTCPADGSGSCDTDALVEDLLRSGSTSPHTVSGGWAAWGPWSSCSRDCELGFRVRKRTCTNPEPRNGGLPCVGDAAEYQDCNPQACPVRGAWSCWTSWSPCSASCGGGHYQRTRSCTSPAPSPGEDICLGLHTEEALCATQACPEGWSPWSEWSKCTDDGAQSRSRHCEELLPGSSACAGNSSQSRPCPYSEIPVILPASSMEEATGCAGFNLIHLVATGISCFLGSGLLTLAVYLSCQHCQRQSQESTLVHPATPNHLHYKGGGTPKNEKYTPMEFKTLNKNNLIPDDRANFYPLQQTNVYTTTYYPSPLNKHSFRPEASPGQRCFPNS(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession no. AY358628);US2003129192 (claim 2); US2004044180 (claim 12); US2004044179 (claim11); US2003096961 (claim 11); US2003232056 (Example 5); WO2003105758(claim 12); US2003206918 (Example 5); EP1347046 (claim 1); WO2003025148(claim 20);

Cross-references: GI:37182378; AAQ88991.1; AY358628_(—)1

141 aa (SEQ ID NO: 8)MWVLGIAATFCGLFLLPGFALQIQCYQCEEFQLNNDCSSPEFIVNCTVNVQDMCQKEVMEQSAGIMYRKSCASSAACLIASAGYQSFCSPGKLNSVCISCCNTPLCNGPRPKKRGSSASALRPGLRTTILFLKLALFSAHC(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177, 34-39, 1991;Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178, 248-255, 1991; AraiH., et al. Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al. J. Biol.Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al. Biochem.Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al. J.Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al. J. Cardiovasc.Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al. Gene 228, 43-49, 1999;Strausberg R. L., et al. Proc. Natl. Acad. Sci. USA. 99, 16899-16903,2002; Bourgeois C., et al. J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al. Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al. Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., etal. Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al.Cell 79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4,2407-2409, 1995; Auricchio A., et al. Hum. Mol. Genet. 5:351-354, 1996;Amiel J., et al. Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., etal. Nat. Genet. 12, 445-447, 1996; Svensson P. J., et al. Hum. Genet.103, 145-148, 1998; Fuchs S., et al. Mol. Med. 7, 115-124, 2001;Pingault V., et al. (2002) Hum. Genet. 111, 198-206; WO2004045516 (claim1); WO2004048938 (Example 2); WO2004040000 (claim 151); WO2003087768(claim 1); WO2003016475 (claim 1); WO2003016475 (claim 1); WO200261087(FIG. 1); WO2003016494 (FIG. 6); WO2003025138 (claim 12; Page 144);WO200198351 (claim 1; Page 124-125); EP522868 (claim 8; FIG. 2);WO200177172 (claim 1; Page 297-299); US2003109676; U.S. Pat. No.6,518,404 (FIG. 3); U.S. Pat. No. 5,773,223 (Claim 1a; Col 31-34);WO2004001004;

442 aa (SEQ ID NO: 9)MQPPPSLCGRALVALVLACGLSRIWGEERGFPPDRATPLLQTAEIMTPPTKTLWPKGSNASLARSLAPAEVPKGDRTAGSPPRTISPPPCQGPIEIKETFKYINTVVSCLVFVLGIIGNSTLLRIIYKNKCMRNGPNILIASLALGDLLHIVIDIPINVYKLLAEDWPFGAEMCKLVPFIQKASVGITVLSLCALSIDRYRAVASWSRIKGIGVPKWTAVEIVLIWVVSVVLAVPEAIGFDIITMDYKGSYLRICLLHPVQKTAFMQFYKTAKDWWLFSFYFCLPLAITAFFYTLMTCEMLRKKSGMQIALNDHLKQRREVAKTVFCLVLVFALCWLPLHLSRILKLTLYNQNDPNRCELLSFLLVLDYIGINMASLNSCINPIALYLVSKRFKNCFKSCLCCWCQSFEEKQSLEEKQSCLKFKANDHGYDNFRSSNKYSSS(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accessionno. NM_(—)017763);WO2003104275 (claim 1); WO2004046342 (Example 2); WO2003042661 (claim12); WO2003083074 (claim 14; Page 61); WO2003018621 (claim 1);WO2003024392 (claim 2; FIG. 93); WO200166689 (Example 6);

Cross-references: LocusID:54894; NP_(—)060233.2; NM_(—)017763_(—)1

783 aa (SEQ ID NO: 10)MSGGHQLQLAALWPWLLMATLQAGFGRTGLVLAAAVESERSAEQKAIIRVIPLKMDPTGKLNLTLEGVFAGVAEITPAEGKLMQSHPLYLCNASDDDNLEPGFISIVKLESPRRAPRPCLSLASKARMAGERGASAVLFDITEDRAAAEQLQQPLGLTWPVVLIWONDAEKLMEFVYKNQKAHVRIELKEPPAWPDYDVWILMTVVGTIFVIILASVLRIRCRPRHSRPDPLQQRTAWAISQLATRRYQASCRQARGEWPDSGSSCSSAPVCAICLEEFSEGQELRVISCLHEFHRNCVDPWLHQHRTCPLCVFNITEGDSFSQSLGPSRSYQEPGRRLHLIRQHPGHAHYHLPAAYLLGPSRSAVARPPRPGPFLPSQEPGMGPRHHRFPRAAHPRAPGEQQRLAGAQHPYAQGWGMSHLQSTSQHPAACPVPLRRARPPDSSGSGESYCTERSGYLADGPASDSSSGPCHGSSSDSVVNCTDISLQGVHGSSSTFCSSLSSDFDPLVYCSPKGDPQRVDMQPSVTSRPRSLDSVVPTGETQVSSHVHYHRHRHHHYKKRFQWHGRKPGPETGVPQSRPPIPRTQPQPEPPSPDQQVTGSNSAAPSGRLSNPQCPRALPEPAPGPVDASSICPSTSSLFNLQKSSLSARHPQRKRRGGPSEPTPGSRPQDATVHPACQIFPHYTPSVAYPWSPEAHPLICGPPGLDKRLLPETPGPCYSNSQPVWLCLTPRQPLEPHPPGEGPSEWSSDTAEGRPCPYPHCQVLSAQPGSEEELEELCE QAV(11) STEAP2 (HGNC_(—)8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP,prostate cancer associated gene 1, prostate cancer associated protein 1,six transmembrane epithelial antigen of prostate 2, six transmembraneprostate protein, Genbank accession no. AF455138, Lab. Invest. 82(11):1573-1582 (2002)); WO2003087306; US2003064397 (claim 1; FIG. 1);WO200272596 (claim 13; Page 54-55); WO200172962 (claim 1; FIG. 4B);WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim22); WO2003042661 (claim 12); US2003060612 (claim 12; FIG. 10);WO200226822 (claim 23; FIG. 2); WO200216429 (claim 12; FIG. 10);

Cross-references: GI:22655488; AAN04080.1; AF455138_(—)1

490 aa (SEQ ID NO: 11)MESISMMGSPKSLSETVLPNGINGIKDARKVIVGVIGSGDFAKSLTIRLIRCGYHVVIGSRNPKFASEFFPHVVDVTHHEDALTKTNIIFVAIHREHYTSLWDLRHLLVGKILIDVSNNMRINQYPESNAEYLASLFPDSLIVKGFNVVSAWALQLGPKDASRQVYICSNNIQARQQVIELARQLNFIPIDLGSLSSAREIENLPLRLFTLWRGPVVVAISLATFFFLYSFVRDVIHPYARNQQSDFYKIPIEIVNKTLPIVAITLLSLVYLAGLLAAAYQLYYGTKYRRFPPWLETWLQCRKQLGLLSFFFAMVHVAYSLCLPMRRSERYLFLNMAYQQVHANIENSWNEEEVWRIEMYISEGIMSLGLLSLLAVTSIPSVSNALNWREFSFIQSTLGYVALLISTFHVLIYGWKRAFEEEYYRFYTPPNFVLALVLPSIVILGKIILFLPCISQKLKRIKKGWEKSQFLEEGIGGTIP HVSPERVTVM(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4, Genbank accession no.NM_(—)017636Xu, X. Z., et al. Proc. Natl. Acad. Sci. USA. 98 (19):10692-10697(2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278 (33):30813-30820(2003)); US2003143557 (claim 4); WO200040614 (claim 14; Page 100-103);WO200210382 (claim 1; FIG. 9A); WO2003042661 (claim 12); WO200230268(claim 27; Page 391); US2003219806 (claim 4); WO200162794 (claim 14;FIG. 1A-D);

Cross-references: MIM:606936; NP_(—)060106.2; NM_(—)017636_(—1)

1214 aa (SEQ ID NO: 12)MVVPEKEQSWIPKIFKKKTCTTFIVDSTDPGGTLCQCGRPRTAHPAVAMEDAFGAAVVTVWDSDAHTTEKPTDAYGELDFTGAGRKHSNFLRLSDRTDPAAVYSLVTRTWGFRAPNLVVSVLGGSGGPVLQTWLQDLLRRGLVRAAQSTGAWIVTGGLHTGIGRHVGVAVRDHQMASTGGTKVVAMGVAPWGVVRNRDTLINPKGSFPARYRWRGDPEDGVQFPLDYNYSAFFLVDDGTHGCLGGENRFRLRLESYISQQKTGVGGTGIDIPVLLLLIDGDEKMLTRIENATQAQLPCLLVAGSGGAADCLAETLEDTLAPGSGGARQGEARDRIRRFFPKGDLEVLQAQVERIMTRKELLTVYSSEDGSEEFETIVLKALVKACGSSEASAYLDELRLAVAWNRVDIAQSELFRGDIQWRSFHLEASLMDALLNDRPEFVRLLISHGLSLGHFLTPMRLAQLYSAAPSNSLIRNLLDQASHSAGTKAPALKGGAAELRPPDVGHVLRMLLGKMCAPRYPSGGAWDPHPGQGFGESMYLLSDKATSPLSLDAGLGQAPWSDLLLWALLLNRAQMAMYFWEMGSNAVSSALGACLLLRVMARLEPDAEEAARRKDLAFKFEGMGVDLFGECYRSSEVRAARLLLRRCPLWGDATCLQLAMQADARAFFAQDGVQSLLTQKWWGDMASTTPIWALVLAFFCPPLIYTRLITFRKSEEEPTREELEFDMDSVINGEGPVGTADPAEKTPLGVPRQSGRPGCCGGRCGGRRCLRRWFHFWGAPVTIFMGNVVSYLLELLLFSRVLLVDEQPAPPGSLELLLYFWAFTLLCEELRQGLSGGGGSLASGGPGPGHASLSQRLRLYLADSWNQCDLVALTCFLLGVGCRLTPGLYHLGRTVLCIDFMVFTVRLLHIFTVNKQLGPKIVIVSKMMKDVFFFLFFLGVWLVAYGVATEGLLRPRDSDFPSILRRVFYRPYLQIFGQIPQEDMDVALMEHSNCSSEPGFWAHPPGAQAGTCVSQYANWLVVLLLVIFLLVANILLVNLLIAMFSYTFGKVQGNSDLYWKAQRYRLIREFHSRPALAPPFIVISHLRLLLRQLCRRPRSPQPSSPALEHFRVYLSKEAERKLLTWESVHKENFLLARARDKRESDSERLKRISQKVDLALKQLGHIREYEQRLKVLEREVQQCSRVLGWVAEALSRSALLPPGGPPPPDLPGSKD(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor, Genbank accession no. NP_(—)003203 or NM_(—)003212,Ciccodicola, A., et al. EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003224411 (claim 1); WO2003083041(Example 1); WO2003034984 (claim 12); WO200288170 (claim 2; Page 52-53);WO2003024392 (claim 2; FIG. 58); WO200216413 (claim 1; Page 94-95, 105);WO200222808 (claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col17-18); U.S. Pat. No. 5,792,616 (FIG. 2);

Cross-references: MIM:187395; NP_(—)003203.1; NM_(—)003212_(—1)

188 aa (SEQ ID NO: 13)MDCRKMARFSYSVIWIMAISKVFELGLVAGLGHQEFARPSRGYLAFRDDSIWPQEEPAIRPRSSQRVPPMGIQHSKELNRTCCLNGGICMLGSFCACPPSFYGRNCEHDVRKENCGSVPHDTWLPKKCSLCKCWHGQLRCFPQAFLPGCDGLVMDEHLVASRTPELPPSARTTTFMLVGI CLSIQSYY(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs.73792 Genbank accession no. M26004,Fujisaku et al. (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., etal. J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al. Proc. Natl.Acad. Sci. USA. 84, 9194-9198, 1987; Barel M., et al. Mol. Immunol. 35,1025-1031, 1998; Weis J. J., et al. Proc. Natl. Acad. Sci. USA. 83,5639-5643, 1986; Sinha S. K., et al. (1993) J. Immunol. 150, 5311-5320;WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (claim9); WO2004045520 (Example 4); WO9102536 (FIG. 9.1-9.9); WO2004020595(claim 1);

Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

1033 aa (SEQ ID NO: 14)MGAAGLLGVFLALVAPGVLGISCGSPPPILNGRISYYSTPIAVGTVIRYSCSGTFRLIGEKSLLCITKDKVDGTWDKPAPKCEYFNKYSSCPEPIVPGGYKIRGSTPYRHGDSVTFACKTNFSMNGNKSVWCQANNMWGPTRLPTCVSVFPLECPALPMIHNGHHTSENVGSIAPGLSVTYSCESGYLLVGEKIINCLSSGKWSAVPPTCEEARCKSLGRFPNGKVKEPPILRVGVTANFFCDEGYRLQGPPSSRCVIAGQGVAWTKMPVCEEIFCPSPPPILNGRHIGNSLANVSYGSIVTYTCDPDPEEGVNFILIGESTLRCTVDSQKTGTWSGPAPRCELSTSAVQCPHPQILRGRMVSGQKDRYTYNDTVIFACMFGFTLKGSKQIRCNAQGTWEPSAPVCEKECQAPPNILNGQKEDRHMVRFDPGTSIKYSCNPGYVLVGEESIQCTSEGVWTPPVPQCKVAACEATGRQLLTKPQHQFVRPDVNSSCGEGYKLSGSVYQECQGTIPWFMEIRLCKEITCPPPPVIYNGAHTGSSLEDFPYGTTVTYTCNPGPERGVEFSLIGESTIRCTSNDQERGTWSGPAPLCKLSLLAVQCSHVHIANGYKISGKEAPYFYNDTVTFKCYSGFTLKGSSQIRCKADNTWDPEIPVCEKETCQHVRQSLQELPAGSRVELVNTSCQDGYQLTGHAYQMCQDAENGIWFKKIPLCKVIHCHPPPVIVNGKHTGMMAENFLYGNEVSYECDQGFYLLGEKKLQCRSDSKGRGSWSGPSPQCLRSPPVTRCPNPEVKHGYKLNKTHSAYSHNDIVYVDCNPGFIMNGSRVIRCHTDNTWVPGVPTCIKKAFIGCPPPPKTPNGNHTGGNIARFSPGMSILYSCDQGYLLVGEALLLCTHEGTWSQPAPHCKEVNCSSPADMDGIQKGLEPRKMYQYGAVVTLECEDGYMLEGSPQSQCQSDHQWNPPLAVCRSRSLAPVLCGIAAGLILLTFLIVITLYVISKHRERNYYTDTSQKEAFHLEAREVYSVDPYNPAS(15) CD79b (CD79B, CD79β, IGb (immunoglobulin-associated beta), B29,Genbank accession no. NM_(—)000626 or 11038674, Proc. Natl. Acad. Sci.USA. (2003) 100 (7):4126-4131, Blood (2002) 100 (9):3068-3076, Muller etal., (1992) Eur. J. Immunol. 22 (6):1621-1625); WO2004016225 (claim 2,FIG. 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401(claim 9); WO200278524 (Example 2); US2002150573 (claim 5, page 15);U.S. Pat. No. 5,644,033; WO2003048202 (claim 1, pages 306 and 309); WO99/558658, U.S. Pat. No. 6,534,482 (claim 13, FIG. 17A/B); WO200055351(claim 11, pages 1145-1146);

Cross-references: MIM:147245; NP_(—)000617.1; NM_(—)000626_(—1)

229 aa (SEQ ID NO: 15)MARLALSPVPSHWMVALLLLLSAEPVPAARSEDRYRNPKGSACSRIWQSPRFIARKRGFTVKMHCYMNSASGNVSWEWKQEMDENPQQLKLEKGRMEESQNESLATLTIQGIRFEDNGIYFCQQKCNNTSEVYQGCGTELRVMGFSTLAQLKQRNTLKDGIIMIQTLLIILFIIVPIFLLLDKDDSKAGMEEDHTYEGLDIDQTATYEDIVTERTGEVKWSVGEHPGQE(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_(—)030764,Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54 (2):87-95(2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci. USA. 98(17):9772-9777 (2001), Xu, M. J., et al. (2001) Biochem. Biophys. Res.Commun. 280 (3):768-775; WO2004016225 (claim 2); WO2003077836;WO200138490 (claim 5; FIG. 18D-1-18D-2); WO2003097803 (claim 12);WO2003089624 (claim 25);

Cross-references: MIM:606509; NP_(—)110391.2; NM_(—)030764_(—)1

508 aa (SEQ ID NO: 16)MLLWSLLVIFDAVTEQADSLTLVAPSSVFEGDSIVLKCQGEQNWKIQKMAYHKDNKELSVFKKFSDFLIQSAVLSDSGNYFCSTKGQLFLWDKTSNIVKIKVQELFQRPVLTASSFQPIEGGPVSLKCETRLSPQRLDVQLQFCFFRENQVLGSGWSSSPELQISAVWSEDTGSYWCKAETVTHRIRKQSLQSQIHVQRIPISNVSLEIRAPGGQVTEGQKLILLCSVAGGTGNVTFSWYREATGTSMGKKTQRSLSAELEIPAVKESDAGKYYCRADNGHVPIQSKVVNIPVRIPVSRPVLTLRSPGAQAAVGDLLELHCEALRGSPPILYQFYHEDVTLGNSSAPSGGGASFNLSLTAEHSGNYSCEANNGLGAQCSEAVPVSISGPDGYRRDLMTAGVLWGLFGVLGFTGVALLLYALFHKISGESSATNEPRGASRPNPQEFTYSSPTPDMEELQPVYVNVGSVDVDVVYSQVWSMQQPESSANIRTLLENKDSQVIYSSVKKS(17) HER2 (ErbB2, Genbank accession no. M11730, Coussens L., et al.Science (1985) 230(4730):1132-1139); Yamamoto T., et al. Nature 319,230-234, 1986; Semba K., et al. Proc. Natl. Acad. Sci. USA. 82,6497-6501, 1985; Swiercz J. M., et al. J. Cell Biol. 165, 869-880, 2004;Kuhns J. J., et al. J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., etal. Nature 421, 756-760, 2003; Ehsani A., et al. (1993) Genomics 15,426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 11); WO2004009622;WO2003081210; WO2003089904 (claim 9); WO2003016475 (claim 1);US2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; FIG.1A-B); WO2003025228 (claim 37; FIG. 5C); WO200222636 (Example 13; Page95-107); WO200212341 (claim 68; FIG. 7); WO200213847 (Page 71-74);WO200214503 (Page 114-117); WO200153463 (claim 2; Page 41-46);WO200141787 (Page 15); WO200044899 (claim 52; FIG. 7); WO200020579(claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (claim 3; Col 31-38);WO9630514 (claim 2; Page 56-61); EP1439393 (claim 7); WO2004043361(claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4); Accession:P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.

1255 aa (SEQ ID NO: 17)MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV(18) NCA (CEACAM6, Genbank accession no. M18728);Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem.Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L., et al. Proc.Natl. Acad. Sci. USA. 99:16899-16903, 2002; WO2004063709; EP1439393(claim 7); WO2004044178 (Example 4); WO2004031238; WO2003042661 (claim12); WO200278524 (Example 2); WO200286443 (claim 27; Page 427);WO200260317 (claim 2);

Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL; M18728;

344 aa (SEQ ID NO: 18)MGPPSAPPCRLHVPWKEVLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLAHNLPQNRIGYSWYKGERVDGNSLIVGYVIGTQQATPGPAYSGRETIYPNASLLIQNVTQNDTGFYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPEVQNTTYLWWVNGQSLPVSPRLQLSNGNMTLTLLSVKRNDAGSYECEIQNPASANRSDPVTLNVLYGPDVPTISPSKANYRPGENLNLSCHAASNPPAQYSWFINGTFQQSTQELFIPNITVNNSGSYMCQAHNSATGLNRTTVTMITVSGSAPVLSAVATVGITIGVLARVALI(19) MDP (DPEP1, Genbank accession no. BC017023,Proc. Natl. Acad. Sci. USA. 99 (26):16899-16903 (2002)); WO2003016475(claim 1); WO200264798 (claim 33; Page 85-87); JP05003790 (FIG. 6-8);WO9946284 (FIG. 9);

Cross-references: MIM:179780; AAH17023.1; BC017023_(—)1

411 aa (SEQ ID NO: 19)MWSGWWLWPLVAVCTADFFRDEAERIMRDSPVIDGHNDLPWQLLDMFNNRLQDERANLTTLAGTHTNIPKLRAGFVGGQFWSVYTPCDTQNKDAVRRTLEQMDVVHRMCRMYPETFLYVTSSAGIRQAFREGKVASLIGVEGGHSIDSSLGVLRALYQLGMRYLTLTHSCNTPWADNWLVDTGDSEPQSQGLSPFGQRVVKELNRLGVLIDLAHVSVATMKATLQLSRAPVIFSHSSAYSVCASRRNVPDDVLRLVKQTDSLVMVNFYNNYISCTNKANLSQVADHLDHIKEVAGARAVGFGGDFDGVPRVPEGLEDVSKYPDLIAELLRRNWTEAEVKGALADNLLRVFEAVEQASNLTQAPEEEPIPLDQLGGSCRTHYGYSSGASSLHRHWGLLLASLAPLVLCLSLL(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971);Clark H. F., et al. Genome Res. 13, 2265-2270, 2003; Mungall A. J., etal. Nature 425, 805-811, 2003; Blumberg H., et al. Cell 104, 9-19, 2001;Dumoutier L., et al. J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J.,et al. J. Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al. (2003)Biochemistry 42:12617-12624; Sheikh F., et al. (2004) J. Immunol. 172,2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);WO2003029262 (Page 74-75); WO2003002717 (claim 2; Page 63); WO200222153(Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59);WO200146232 (Page 63-65); WO9837193 (claim 1; Page 55-59);

Accession: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

553 aa (SEQ ID NO: 20)MRAPGRPALRPLPLPPLLLLLLAAPWGRAVPCVSGGLPKPANITFLSINMKNVLQWTPPEGLQGVKVTYTVQYFIYGQKKWLNKSECRNINRTYCDLSAETSDYEHQYYAKVKAIWGTKCSKWAESGRFYPFLETQIGPPEVALTTDEKSISVVLTAPEKWKRNPEDLPVSMQQTYSNLKYNVSVLNTKSNRTWSQCVTNHTLVLTWLEPNTLYCVHVESFVPGPPRRAQPSEKQCARTLKDQSSEFKAKIIFWYVLPISITVFLFSVMGYSIYRYIHVGKEKHPANLILIYGNEFDKRFFVPAEKIVINFITLNISDDSKISHQDMSLLGKSSDVSSLNDPQPSGNLRPPQEEEEVKHLGYASHLMEIFCDSEENTEGTSFTQQESLSRTIPPDKTVIEYEYDVRTTDICAGPEEQELSLQEEVSTQGTLLESQAALAVLGPQTLQYSYTPQLQDLDPLAQEHTDSEEGPEEEPSTTLVDWDPQTGRLCIPSLSSFDQDSEGCEPSEGDGLGEEGLLSRLYEEPAPDRPPGENETYLMQFMEEWGLYVQMEN(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053)Gary S. C., et al. Gene 256, 139-147, 2000; Clark H. F., et al. GenomeRes. 13, 2265-2270, 2003; Strausberg R. L., et al. Proc. Natl. Acad.Sci. USA. 99, 16899-16903, 2002; US2003186372 (claim 11); US2003186373(claim 11); US2003119131 (claim 1; FIG. 52); US2003119122 (claim 1; FIG.52); US2003119126 (claim 1); US2003119121 (claim 1; FIG. 52);US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1;FIG. 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634(claim 1);

911 aa (SEQ ID NO: 21)MAQLFLPLLAALVLAQAPAALADVLEGDSSEDRAFRVRIAGDAPLQGVLGGALTIPCHVHYLRPPPSRRAVLGSPRVKWTFLSRGREAEVLVARGVRVKVNEAYRFRVALPAYPASLTDVSLALSELRPNDSGIYRCEVQHGIDDSSDAVEVKVKGVVFLYREGSARYAFSFSGAQEACARIGAHIATPEQLYAAYLGGYEQCDAGWLSDQTVRYPIQTPREACYGDMDGFPGVRNYGVVDPDDLYDVYCYAEDLNGELFLGDPPEKLTLEEARAYCQERGAEIATTGQLYAAWDGGLDHCSPGWLADGSVRYPIVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRFNVYCFRDSAQPSAIPEASNPASNPASDGLEAIVTVTETLEELQLPQEATESESRGAIYSIPIMEDGGGGSSTPEDPAEAPRTLLEFETQSMVPPTGFSEEEGKALEEEEKYEDEEEKEEEEEEEEVEDEALWAWPSELSSPGPEASLPTEPAAQEKSLSQAPARAVLQPGASPLPDGESEASRPPRVHGPPTETLPTPRERNLASPSPSTLVEAREVGEATGGPELSGVPRGESEETGSSEGAPSLLPATRAPEGTRELEAPSEDNSGRTAPAGTSVQAQPVLPTDSASRGGVAVVPASGDCVPSPCHNGGTCLEEEEGVRCLCLPGYGGDLCDVGLRFCNPGWDAFQGACYKHFSTRRSWEEAETQCRMYGAHLASISTPEEQDFINNRYREYQWIGLNDRTIEGDFLWSDGVPLLYENWNPGQPDSYFLSGENCVVMVWHDQGQWSDVPCNYHLSYTCKMGLVSCGPPPELPLAQVFGRPRLRYEVDTVLRYRCREGLAQRNLPLIRCQENGRWEAPQISCVPRRPARALHPEEDPEGRQGRLLGRWKAL LIPPSSPMPGP(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no.NM_(—)004442) Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991)Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998),Int. Rev. Cytol. 196:177-244 (2000)); WO2003042661 (claim 12);WO200053216 (claim 1; Page 41); WO2004065576 (claim 1); WO2004020583(claim 9); WO2003004529 (Page 128-132); WO200053216 (claim 1; Page 42);

Cross-references: MIM:600997; NP_(—)004433.2; NM_(—)004442_(—)1

987 aa (SEQ ID NO: 22)MALRRLGAALLLLPLLAAVEETLMDSTTATAELGWMVHPPSGWEEVSGYDENMNTIRTYQVCNVFESSQNNWLRTKFIRRRGAHRIHVEMKFSVRDCSSIPSVPGSCKETFNLYYYEADFDSATKTFPNWMENPWVKVDTIAADESFSQVDLGGRVMKINTEVRSEGPVSRSGFYLAFQDYGGCMSLIAVRVFYRKCPRIIQNGAIFQETLSGAESTSLVAARGSCIANAEEVDVPIKLYCNGDGEWLVPIGRCMCKAGFEAVENGTVCRGCPSGTFKANQGDEACTHCPINSRTTSEGATNCVCRNGYYRADLDPLDMPCTTIPSAPQAVISSVNETSLMLEWTPPRDSGGREDLVYNIICKSCGSGRGACTRCGDNVQYAPRQLGLTEPRIYISDLLAHTQYTFEIQAVNGVTDQSPFSPQFASVNITTNQAAPSAVSIMHQVSRTVDSITLSWSQPDQPNGVILDYELQYYEKELSEYNATAIKSPTNTVTVQGLKAGAIYVFQVRARTVAGYGRYSGKMYFQTMTEAEYQTSIQEKLPLIIGSSAAGLVFLIAVVVIAIVCNRRRGFERADSEYTDKLQHYTSGHMTPGMKIYIDPFTYEDPNEAVREFAKEIDISCVKIEQVIGAGEFGEVCSGHLKLPGKREIEVAIKTLKSGYTEKQRRDFLSEASIMGQFDHPNVIHLEGVVTKSTPVMIITEFMENGSLDSFLRQNDGQFTVIQLVGMLRGIAAGMKYLADMNYVHRDLAARNILVNSNLVCKVSDFGLSRFLEDDTSDPTYTSALGGKIPIRWTAPEAIQYRKFTSASDVWSYGIVMWEVMSYGERPYWDMTNQDVINAIEQDYRLPPPMDCPSALHQLMLDCWQKDRNHRPKFGQIVNTLDKMIRNPNSLKAMAPLSSGINLPLLDRTIPDYTSFNTVDEWLEAIKMGQYKESFANAGFTSFDVVSQMMMEDILRVGVTLAGHQKKILNSIQVMRAQMNQIQSVEV(23) ASLG659 (B7h, Genbank accession no. AX092328)US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (FIG. 3);US2003165504 (claim 1); US2003124140 (Example 2); US2003065143 (FIG.60); WO2002102235 (claim 13; Page 299); US2003091580 (Example 2);WO200210187 (claim 6; FIG. 10); WO200194641 (claim 12; FIG. 7 b);WO200202624 (claim 13; FIG. 1A-1B); US2002034749 (claim 54; Page 45-46);WO200206317 (Example 2; Page 320-321, claim 34; Page 321-322);WO200271928 (Page 468-469); WO200202587 (Example 1; FIG. 1); WO200140269(Example 3; Pages 190-192); WO200036107 (Example 2; Page 205-207);WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (Page233-234, 452-453); WO 0116318;

282 aa (SEQ ID NO: 23)MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKKNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK(24) PSCA (Prostate stem cell antigen precursor, Genbank accession no.AJ297436) Reiter R. E., et al. Proc. Natl. Acad. Sci. USA. 95,1735-1740, 1998; Gu Z., et al. Oncogene 19, 1288-1296, 2000; Biochem.Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274(Example 11); US2004018553 (claim 17); WO2003008537 (claim 1);WO200281646 (claim 1; Page 164); WO2003003906 (claim 10; Page 288);WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; FIG. 1 b);WO200032752 (claim 18; FIG. 1); WO9851805 (claim 17; Page 97); WO9851824(claim 10; Page 94); WO9840403 (claim 2; FIG. 1B);

Accession: O43653; EMBL; AF043498; AAC39607.1.

123 aa MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQL(25) GEDA (Genbank accession No. AY260763);AAP14954 lipoma HMGIC fusion-partner-like protein/pid=AAP14954.1-Homosapiens Species: Homo sapiens (human)WO2003054152 (claim 20); WO2003000842 (claim 1); WO2003023013 (Example3, claim 20); US2003194704 (claim 45);

Cross-references: GI:30102449; AAP14954.1; AY260763_(—)1

236 aa (SEQ ID NO: 25)MPGAAAAAAAAAAAMLPAQEAAKLYHTNYVRNSRAIGVLWAIFTICFAIVNVVCFIQPYWIGDGVDTPQAGYFGLFHYCIGNGFSRELTCRGSFTDFSTLPSGAFKAASFFIGLSMMLIIACIICFTLFFFCNTATVYKICAWMQLTSAACLVLGCMIFPDGWDSDEVKRMCGEKTDKYTLGACSVRWAYILAIIGILDALILSFLAFVLGNRQDSLMAEELKAENKVLLSQYSLE(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3,Genbank accession No. NP_(—)443177.1);NP_(—)443177 BAFF receptor/pid=NP_(—)443177.1-Homo sapiensThompson, J. S., et al. Science 293 (5537), 2108-2111 (2001);WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33);WO2003014294 (claim 35; FIG. 6B); WO2003035846 (claim 70; Page 615-616);WO200294852 (Col 136-137); WO200238766 (claim 3; Page 133); WO200224909(Example 3; FIG. 3);

Cross-references: MIM:606269; NP_(—)443177.1; NM_(—)052945_(—)1

184 aa (SEQ ID NO: 26)MRRGPRSLRGRDAPAPTPCVPAECFDLLVRHCVACGLLRTPRPKPAGASSPAPRTALQPQESVGAGAGEAALPLPGLLFGAPALLGLALVLALVLVGLVSWRRRQRRLRGASSAEAPDGDKDAPEPLDKVIILSPGISDATAPAWPPPGEDPGTTPPGHSVPVPATELGSTELVTTKTAGPEQQ(27) CD22 (B-cell receptor CD22-β isoform, Genbank accession No.NP-001762.1);Stamenkovic, I. and Seed, B., Nature 345 (6270), 74-77 (1990);US2003157113; US2003118592; WO2003062401 (claim 9); WO2003072036 (claim1; FIG. 1); WO200278524 (Example 2);

Cross-references: MIM:107266; NP 001762.1; NM_(—)001771_(—)1

847 aa (SEQ ID NO: 27)MHLLGPWLLLLVLEYLAFSDSSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFHNPEYNKNTSKFDGTRLYESTKDGKVPSEQKRVQFLGDKNKNCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSERPFPPHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIKSVFTRSELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTPSDAIVREGDSVTMTCEVSSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVTKDQSGKYCCQVSNDVGPGRSEEVFLQVQYAPEPSTVQILHSPAVEGSQVEFLCMSLANPLPTNYTWYHNGKEMQGRTEEKVHIPKILPWHAGTYSCVAENILGTGQRGPGAELDVQYPPKKVTTVIQNPMPIREGDTVTLSCNYNSSNPSVTRYEWKPHGAWEEPSLGVLKIQNVGWDNTTIACARCNSWCSWASPVALNVQYAPRDVRVRKIKPLSEIHSGNSVSLQCDFSSSHPKEVQFFWEKNGRLLGKESQLNFDSISPEDAGSYSCWVNNSIGQTASKAWTLEVLYAPRRLRVSMSPGDQVMEGKSATLTCESDANPPVSHYTWFDWNNQSLPHHSQKLRLEPVKVQHSGAYWCQGTNSVGKGRSPLSTLTVYYSPETIGRRVAVGLGSCLAILILAICGLKLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTGDAESSEMQRPPRTCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELIQFGVGERPQAQENVDYVILKH(28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation) PROTEIN SEQUENCE Full mpggpgv . . .dvqlekp (1 . . . 226; 226 aa), pI: 4.84, MW: 25028 TM: 2 [P] GeneChromosome: 19q13.2, Genbank accession No. NP_(—)001774.1;WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim4, pages 13-14); WO9958658 (claim 13, FIG. 16); WO9207574 (FIG. 1); U.S.Pat. No. 5,644,033; Ha et al. (1992) J. Immunol. 148(5):1526-1531;Mueller et al. (1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al.(1994) Immunogenetics 40(4):287-295; Preud'homme et al. (1992) Clin.Exp. Immunol. 90(1):141-146; Yu et al. (1992) J. Immunol. 148(2)633-637; Sakaguchi et al. (1988) EMBO J. 7(11):3457-3464;

226 aa (SEQ ID NO: 28)MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGEDAHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEGIILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia) PROTEINSEQUENCE Full mnypltl . . . atslttf (1 . . . 372; 372 aa), pI: 8.54 MW:41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accession No.NP_(—)001707.1; WO2004040000; WO2004015426; US2003105292 (Example 2);U.S. Pat. No. 6,555,339 (Example 2); WO200261087 (FIG. 1); WO200157188(claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (Example 1,pages 152-153, Example 2, pages 254-256); WO9928468 (claim 1, page 38);U.S. Pat. No. 5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58);WO9217497 (claim 7, FIG. 5); Dobner et al., (1992) Eur. J. Immunol.22:2795-2799; Barella et al. (1995) Biochem. J. 309:773-779;

372 aa (SEQ ID NO: 29)MNYPLTLEMDLENLEDLFWELDRLDNYNDTSLVENHLCPATEGPLMASFKAVFVPVAYSLIFLLGVIGNVLVLVILERHRQTRSSTETFLFHLAVADLLLVFILPFAVAEGSVGWVLGTFLCKTVIALHKVNFYCSSLLLACIAVDRYLAIVHAVHAYRHRRLLSIHITCGTIWLVGFLLALPEILFAKVSQGHHNNSLPRCTFSQENQAETHAWFTSRFLYHVAGFLLPMLVMGWCYVGVVHRLRQAQRRPQRQKAVRVAILVTSIFFLCWSPYHIVIFLDTLARLKAVDNTCKLNGSLPVAITMCEFLGLAHCCLNPMLYTFAGVKFRSDLSRLLTKLGCTGPASLCQLFPSWRRSSLSESENATSLTTF(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+ T lymphocytes) PROTEIN SEQUENCEFull mgsgwvp . . . vllpqsc (1 . . . 273; 273 aa, pI: 6.56 MW: 30820 TM:1 [P] Gene Chromosome: 6p21.3, Genbank accession No. NP_(—)002111.1;Tonnelle et al. (1985) EMBO J. 4(11):2839-2847; Jonsson et al. (1989)Immunogenetics 29(6):411-413; Beck et al. (1992) J. Mol. Biol.228:433-441; Strausberg et al. (2002) Proc. Natl. Acad. Sci. USA99:16899-16903; Servenius et al. (1987) J. Biol. Chem. 262:8759-8766;Beck et al. (1996) J. Mol. Biol. 255:1-13; Naruse et al. (2002) TissueAntigens 59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No.6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat.No. 6,011,146 (col 145-146); Kasahara et al. (1989) Immunogenetics30(1):66-68; Larhammar et al. (1985) J. Biol. Chem. 260(26):14111-14119;

273 aa (SEQ ID NO: 30)MGSGWVPWVVALLVNLTRLDSSMTQGTDSPEDFVIQAKADCYFTNGTEKVQFVVRFIFNLEEYVRFDSDVGMFVALTKLGQPDAEQWNSRLDLLERSRQAVDGVCRHNYRLGAPFTVGRKVQPEVTVYPERTPLLHQHNLLHCSVTGFYPGDIKIKWFLNGQEERAGVMSTGPIRNGDWTFQTVVMLEMTPELGHVYTCLVDHSSLLSPVSVEWRAQSEYSWRKMLSGIAAFLLGLIFLLVGIVIQLRAQKGYVRTQMSGNEVSRAVLLPQSC(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability) PROTEIN SEQUENCEFull mgqagck . . . lephrst (1.422; 422 aa), pl: 7.63, MW: 47206 TM: 1[P] Gene Chromosome: 17p13.3, Genbank accession No. NP_(—)002552.2;Le et al. (1997) FEBS Lett. 418(1-2):195-199; WO2004047749; WO2003072035(claim 10); Touchman et al. (2000) Genome Res. 10:165-173; WO200222660(claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277(page 82);

422 aa MGQAGCKGLCLSLFDYKTEKYVIAKNKKVGLLYRLLQASILAYLVVWVFLIKKGYQDVDTSLQSAVITKVKGVAFTNTSDLGQRIWDVADYVIPAQGENVFFVVTNLIVTPNQRQNVCAENEGIPDGACSKDSDCHAGEAVTAGNGVKTGRCLRRENLARGTCEIFAWCPLETSSRPEEPFLKEAEDFTIFIKNHIRFPKFNFSKSNVMDVKDRSFLKSCHFGPKNHYCPIFRLGSVIRWAGSDFQDIALEGGVIGINIEWNCDLDKAASECHPHYSFSRLDNKLSKSVSSGYNFRFARYYRDAAGVEFRTLMKAYGIRFDVMVNGKGAFFCDLVLIYLIKKREFYRDKKYEEVRGLEDSSQEAEDEASGLGLSEQLTSGPGLLGMPEQQELQEPPEAKRGSSSQKGNGSVCPQLLEPHRST(32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCEFull maeaity . . . tafrfpd (1.359; 359 aa), pI: 8.66, MW: 40225 TM: 1[P] Gene Chromosome: 9p13.3, Genbank accession No. NP_(—)001773.1;WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655(pages 105-106); Von Hoegen et al. (1990) J. Immunol. 144(12):4870-4877;Strausberg et al. (2002) Proc. Natl. Acad. Sci. USA 99:16899-16903;

359 aa (SEQ ID NO: 32)MAEAITYADLRFVKAPLKKSISSRLGQDPGADDDGEITYENVQVPAVLGVPSSLASSVLGDKAAVKSEQPTASWRAVTSPAVGRILPCRTTCLRYLLLGLLLTCLLLGVTAICLGVRYLQVSQQLQQTNRVLEVTNSSLRQQLRLKITQLGQSAEDLQGSRRELAQSQEALQVEQRAHQAAEGQLQACQADRQKTKETLQSEEQQRRALEQKLSNMENRLKPFFTCGSADTCCPSGWIMHQKSCFYISLTSKNWQESQKQCETLSSKLATESEIYPQSHSYYFLNSLLPNGGSGNSYWTGLSSNKDWKLTDDTQRTRTYAQSSKCNKVHKTWSWWTLESESCRSSLPYIC EMTAFRFPD(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis) PROTEIN SEQUENCEFull mafdvsc . . . rwkyghi (1 . . . 661; 661 aa), pI: 6.20, MW: 74147TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No. NP_(—)005573.1;US2002193567; WO9707198 (claim 11, pages 39-42); Miura et al. (1996)Genomics 38(3):299-304; Miura et al. (1998) Blood 92:2815-2822;WO2003083047; WO9744452 (claim 8, pages 57-61); WO200012130 (pages24-26);

661 aa (SEQ ID NO: 33)MAFDVSCFFWVVLFSAGCKVITSWDQMCIEKEANKTYNCENLGLSEIPDTLPNTTEFLEFSFNFLPTIHNRTFSRLMNLTFLDLTRCQINWIHEDTFQSHHQLSTLVLTGNPLIFMAETSLNGPKSLKHLFLIQTGISNLEFIPVHNLENLESLYLGSNHISSIKFPKDFPARNLKVLDFQNNAIHYISREDMRSLEQAINLSLNFNGNNVKGIELGAFDSTVFQSLNFGGTPNLSVIFNGLQNSTTQSLWLGTFEDIDDEDISSAMLKGLCEMSVESLNLQEHRFSDISSTTFQCFTQLQELDLTATHLKGLPSGMKGLNLLKKLVLSVNHFDQLCQISAANFPSLTHLYIRGNVKKLHLGVGCLEKLGNLQTLDLSHNDIEASDCCSLQLKNLSHLQTLNLSHNEPLGLQSQAFKECPQLELLDLAFTRLHINAPQSPFQNLHFLQVLNLTYCFLDTSNQHLLAGLPVLRHLNLKGNHFQDGTITKTNLLQTVGSLEVLILSSCGLLSIDQQAFHSLGKMSHVDLSHNSLTCDSIDSLSHLKGIYLNLAANSINIISPRLLPILSQQSTINLSHNPLDCTCSNIHFLTWYKENLHKLEGSEETTCANPPSLRGVKLSDVKLSCGITAIGIFFLIVFLLLLAILLFFAV KYLLRWKYQHI(34) FCRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation) PROTEIN SEQUENCE Fullmlprlll . . . vdyedam (1 . . . 429; 429 aa), pI: 5.28, MW: 46925 TM: 1[P] Gene Chromosome: 1q21-1q22, Genbank accession No. NP 443170.1;WO2003077836; WO200138490 (claim 6, FIG. 18E-1-18-E-2); Davis et al.(2001) Proc. Natl. Acad. Sci. USA 98(17):9772-9777; WO2003089624 (claim8); EP1347046 (claim 1); WO2003089624 (claim 7);

429 aa (SEQ ID NO: 34)MLPRLLLLICAPLCEPAELFLIASPSHPTEGSPVTLTCKMPFLQSSDAQFQFCFFRDTRALGPGWSSSPKLQIAAMWKEDTGSYWCEAQTMASKVLRSRRSQINVHRVPVADVSLETQPPGGQVMEGDRLVLICSVAMGTGDITFLWYKGAVGLNLQSKTQRSLTAEYEIPSVRESDAEQYYCVAENGYGPSPSGLVSITVRIPVSRPILMLRAPRAQAAVEDVLELHCEALRGSPPILYWFYHEDITLGSRSAPSGGGASFNLSLTEEHSGNYSCEANNGLGAQRSEAVTLNFTVPTGARSNHLTSGVIEGLLSTLGPATVALLFCYGLKRKIGRRSARDPLRSLPSPLPQEFTYLNSPTPGQLQPIYENVNVVSGDEVYSLAYYNQPEQESVAAETLGTHMEDKVSLDIYSRLRKANITDVDYEDAM (35) IRTA2 (Immunoglobulin superfamily receptor translocation associated2, a putative immunoreceptor with possible roles in B cell developmentand lymphomagenesis; deregulation of the gene by translocation occurs insome B cell malignancies) PROTEIN SEQUENCE Full mllwvil . . . assaphr (1. . . 977; 977 aa), pI: 6.88 MW: 106468 TM: 1 [P] Gene Chromosome: 1q21,Genbank accession No. NP_(—)112571.1;WO2003024392 (claim 2, FIG. 97); Nakayama et al. (2000) Biochem.Biophys. Res. Commun. 277(1):124-127; WO2003077836; WO200138490 (claim3, FIG. 18B-1-18B-2);

977 aa (SEQ ID NO: 35)MLLWVILLVLAPVSGQFARTPRPIIFLQPPWTTVFQGERVTLTCKGFRFYSPQKTKWYHRYLGKEILRETPDNILEVQESGEYRCQAQGSPLSSPVHLDFSSASLILQAPLSVFEGDSVVLRCRAKAEVTLNNTIYKNDNVLAFLNKRTDFHIPHACLKDNGAYRCTGYKESCCPVSSNTVKIQVQEPFTRPVLRASSFQPISGNPVTLTCETQLSLERSDVPLRFRFFRDDQTLGLGWSLSPNFQITAMWSKDSGFYWCKAATMPHSVISDSPRSWIQVQIPASHPVLTLSPEKALNFEGTKVTLHCETQEDSLRTLYRFYHEGVPLRHKSVRCERGASISFSLTTENSGNYYCTADNGLGAKPSKAVSLSVTVPVSHPVLNLSSPEDLIFEGAKVTLHCEAQRGSLPILYQFHHEDAALERRSANSAGGVAISFSLTAEHSGNYYCTADNGFGPQRSKAVSLSITVPVSHPVLTLSSAEALTFEGATVTLHCEVQRGSPQILYQFYHEDMPLWSSSTPSVGRVSFSFSLTEGHSGNYYCTADNGFGPQRSEVVSLFVTVPVSRPILTLRVPRAQAVVGDLLELHCEAPRGSPPILYWFYHEDVTLGSSSAPSGGEASFNLSLTAEHSGNYSCEANNGLVAQHSDTISLSVIVPVSRPILTFRAPRAQAVVGDLLELHCEALRGSSPILYWFYHEDVTLGKISAPSGGGASFNLSLTTEHSGIYSCEADNGPEAQRSEMVTLKVAVPVSRPVLTLRAPGTHAAVGDLLELHCEALRGSPLILYRFFHEDVTLGNRSSPSGGASLNLSLTAEHSGNYSCEADNGLGAQRSETVTLYITGLTANRSGPFATGVAGGLLSIAGLAAGALLLYCWLSRKAGRKPASDPARSPPDSDSQEPTYHNVPAWEELQPVYTNANPRGENVVYSEVRIIQEKKKHAVASDPRHLRNKGSPIIYSEVKVASTPVSGSLFLASSAPHR

See also: WO04/045516 (3 Jun. 2004); WO03/000113 (3 Jan. 2003);WO02/016429 (28 Feb. 2002); WO02/16581 (28 Feb. 2002); WO03/024392 (27Mar. 2003); WO04/016225 (26 Feb. 2004); WO01/40309 (7 Jun. 2001), andU.S. Provisional patent application Ser. No. 60/520,842 “COMPOSITIONSAND METHODS FOR THE TREATMENT OF TUMOR OF HEMATOPOIETIC ORIGIN”, filed17 Nov. 2003; all of which are incorporated herein by reference in theirentirety.

In an embodiment, the Ligand-Linker-Drug Conjugate has Formula IIIa,where the Ligand is an antibody Ab including one that binds at least oneof CD30, CD40, CD70, Lewis Y antigen, w=0, y=0, and D has Formula Ib.Exemplary Conjugates of Formula IIIa include where R¹⁷ is —(CH₂)₅—. Alsoincluded are such Conjugates of Formula IIIa in which D has thestructure of Compound 2 in Example 3 and esters thereof. Also includedare such Conjugates of Formula Ma containing about 3 to about 8, in oneaspect, about 3 to about 5 Drug moieties D, that is, Conjugates ofFormula Ia wherein p is a value in the range about 3-8, for exampleabout 3-5. Conjugates containing combinations of the structural featuresnoted in this paragraph are also contemplated as within the scope of thecompounds of the invention.

In another embodiment, the Ligand-Linker-Drug Conjugate has FormulaIIIa, where Ligand is an Antibody Ab that binds one of CD30, CD40, CD70,Lewis Y antigen, w=1, y=0, and D has Formula Ib. Included are suchConjugates of Formula IIIa in which R¹⁷ is —(CH₂)₅—. Also included aresuch Conjugates of Formula IIIa in which W is -Val-Cit-, and/or where Dhas the structure of Compound 2 in Example 3 and esters thereof. Alsoincluded are such Conjugates of Formula IIIa containing about 3 to about8, preferably about 3 to about 5 Drug moieties D, that is, Conjugates ofFormula Ia wherein p is a value in the range of about 3-8, preferablyabout 3-5. Conjugates containing combinations of the structural featuresnoted in this paragraph are also exemplary.

In an embodiment, the Ligand-Linker-Drug Conjugate has Formula IIIa,where the Ligand is an Antibody Ab that binds one of CD30, CD40, CD70,Lewis Y antigen, w=1, y=1, and D has Formula Ib. Included are Conjugatesof Formula Ina in which R¹⁷ is —(CH₂)₅—. Also included are suchConjugates of Formula IIIa where: W is -Val-Cit-; Y has Formula X; D hasthe structure of Compound 2 in Example 3 and esters thereof; p is about3 to about 8, preferably about 3 to about 5 Drug moieties D. Conjugatescontaining combinations of the structural features noted in thisparagraph are also contemplated within the scope of the compounds of theinvention.

A further embodiment is an antibody drug conjugate (ADC), or apharmaceutically acceptable salt or solvate thereof, wherein Ab is anantibody that binds one of the tumor-associated antigens (1)-(35) notedabove (the “TAA Compound”).

Another embodiment is the TAA Compound or pharmaceutically acceptablesalt or solvate thereof that is in isolated and purified form.

Another embodiment is a method for killing or inhibiting themultiplication of a tumor cell or cancer cell comprising administeringto a patient, for example a human with a hyperproliferative disorder, anamount of the TAA Compound or a pharmaceutically acceptable salt orsolvate thereof, said amount being effective to kill or inhibit themultiplication of a tumor cell or cancer cell.

Another embodiment is a method for treating cancer comprisingadministering to a patient, for example a human with ahyperproliferative disorder, an amount of the TAA Compound or apharmaceutically acceptable salt or solvate thereof, said amount beingeffective to treat cancer, alone or together with an effective amount ofan additional anticancer agent.

Another embodiment is a method for treating an autoimmune disease,comprising administering to a patient, for example a human with ahyperproliferative disorder, an amount of the TAA Compound or apharmaceutically acceptable salt or solvate thereof, said amount beingeffective to treat an autoimmune disease.

The antibodies suitable for use in the invention can be produced by anymethod known in the art for the synthesis of antibodies, in particular,by chemical synthesis or by recombinant expression, and are preferablyproduced by recombinant expression techniques.

Production of Recombinant Antibodies

Antibodies of the invention can be produced using any method known inthe art to be useful for the synthesis of antibodies, in particular, bychemical synthesis or by recombinant expression.

Recombinant expression of antibodies, or fragment, derivative or analogthereof, requires construction of a nucleic acid that encodes theantibody. If the nucleotide sequence of the antibody is known, a nucleicacid encoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., 1994,BioTechniques 17:242), which involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides, e.g., by PCR.

Alternatively, a nucleic acid molecule encoding an antibody can begenerated from a suitable source. If a clone containing the nucleic acidencoding the particular antibody is not available, but the sequence ofthe antibody is known, a nucleic acid encoding the antibody can beobtained from a suitable source (e.g., an antibody cDNA library, or cDNAlibrary generated from any tissue or cells expressing theimmunoglobulin) by, e.g., PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence.

If an antibody that specifically recognizes a particular antigen is notcommercially available (or a source for a cDNA library for cloning anucleic acid encoding such an immunoglobulin), antibodies specific for aparticular antigen can be generated by any method known in the art, forexample, by immunizing a patient, or suitable animal model such as arabbit or mouse, to generate polyclonal antibodies or, more preferably,by generating monoclonal antibodies, e.g., as described by Kohler andMilstein (1975, Nature 256:495-497) or, as described by Kozbor et al.(1983, Immunology Today 4:72) or Cole et al. (1985 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).Alternatively, a clone encoding at least the Fab portion of the antibodycan be obtained by screening Fab expression libraries (e.g., asdescribed in Huse et al., 1989, Science 246:1275-1281) for clones of Fabfragments that bind the specific antigen or by screening antibodylibraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane etal., 1997 Proc. Natl. Acad. Sci. USA 94:4937).

Once a nucleic acid sequence encoding at least the variable domain ofthe antibody is obtained, it can be introduced into a vector containingthe nucleotide sequence encoding the constant regions of the antibody(see, e.g., International Publication No. WO 86/05807; WO 89/01036; andU.S. Pat. No. 5,122,464). Vectors containing the complete light or heavychain that allow for the expression of a complete antibody molecule areavailable. Then, the nucleic acid encoding the antibody can be used tointroduce the nucleotide substitutions or deletion necessary tosubstitute (or delete) the one or more variable region cysteine residuesparticipating in an intrachain disulfide bond with an amino acid residuethat does not contain a sulfhydyl group. Such modifications can becarried out by any method known in the art for the introduction ofspecific mutations or deletions in a nucleotide sequence, for example,but not limited to, chemical mutagenesis and in vitro site directedmutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551).

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855;Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature314:452-454) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-54) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,1988, Science 242:1038-1041).

Antibody fragments that recognize specific epitopes can be generated byknown techniques. For example, such fragments include, but are notlimited to the F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule and the Fab fragments that can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.

Once a nucleic acid sequence encoding an antibody has been obtained, thevector for the production of the antibody can be produced by recombinantDNA technology using techniques well known in the art. Methods that arewell known to those skilled in the art can be used to constructexpression vectors containing the antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. See, forexample, the techniques described in Sambrook et al. (1990, MolecularCloning, A Laboratory Manual, 2^(nd) Éd., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.) and Ausubel et al. (eds., 1998, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY).

An expression vector comprising the nucleotide sequence of an antibodyor the nucleotide sequence of an antibody can be transferred to a hostcell by conventional techniques (e.g., electroporation, liposomaltransfection, and calcium phosphate precipitation), and the transfectedcells are then cultured by conventional techniques to produce theantibody. In specific embodiments, the expression of the antibody isregulated by a constitutive, an inducible or a tissue, specificpromoter.

The host cells used to express the recombinant antibody can be eitherbacterial cells such as Escherichia coli, or, preferably, eukaryoticcells, especially for the expression of whole recombinant immunoglobulinmolecule. In particular, mammalian cells such as Chinese hamster ovarycells (CHO), in conjunction with a vector such as the major intermediateearly gene promoter element from human cytomegalovirus is an effectiveexpression system for immunoglobulins (Foecking et al., 198, Gene45:101; Cockett et al., 1990, BioTechnology 8:2).

A variety of host-expression vector systems can be utilized to expressthe immunoglobulin antibodies. Such host-expression systems representvehicles by which the coding sequences of the antibody can be producedand subsequently purified, but also represent cells that can, whentransformed or transfected with the appropriate nucleotide codingsequences, express an antibody immunoglobulin molecule in situ. Theseinclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing immunoglobulincoding sequences; yeast (e.g., Saccharomyces Pichia) transformed withrecombinant yeast expression vectors containing immunoglobulin codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the immunoglobulincoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus (CaMV) and tobaccomosaic virus (TMV)) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing immunoglobulin coding sequences;or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such a protein isto be produced, vectors that direct the expression of high levels offusion protein products that are readily purified might be desirable.Such vectors include, but are not limited, to the E. coli expressionvector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which theantibody coding sequence may be ligated individually into the vector inframe with the lac Z coding region so that a fusion protein is produced;pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEXVectors can also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption and binding to a matrix glutathione-agarose beads followed byelution in the presence of free glutathione. The pGEX vectors aredesigned to include thrombin or factor Xa protease cleavage sites sothat the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa califormica nuclear polyhedrosis virus(AcNPV) or the analogous virus from Drosophila Melanogaster is used as avector to express foreign genes. The virus grows in Spodopterafrugiperda cells. The antibody coding sequence can be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) results in a recombinant virus that is viable and capable ofexpressing the immunoglobulin molecule in infected hosts. (e.g., seeLogan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specificinitiation signals can also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., 1987,Methods in Enzymol. 153:51-544).

In addition, a host cell strain can be chosen to modulate the expressionof the inserted sequences, or modifies and processes the gene product inthe specific fashion desired. Such modifications (e.g., glycosylation)and processing (e.g., cleavage) of protein products can be important forthe function of the protein. Different host cells have characteristicand specific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification andprocessing of the foreign protein expressed. To this end, eukaryotichost cells that possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include, but are notlimited to, CHO, VERY, BH, Hela, COS, MDCK, 293, 293T, 3T3, WI38, BT483,Hs578T, HTB2, BT20 and T47D, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express anantibody can be engineered. Rather than using expression vectors thatcontain viral origins of replication, host cells can be transformed withDNA controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells can be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci that in turn can becloned and expanded into cell lines. This method can advantageously beused to engineer cell lines which express the antibody. Such engineeredcell lines can be particularly useful in screening and evaluation oftumor antigens that interact directly or indirectly with the antibody.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: DHFR, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993, TIB TECH 11(5):155-215) and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds., 1993, CurrentProtocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1).

The expression levels of an antibody can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing an antibody isamplifiable, an increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the nucleotide sequence of theantibody, production of the antibody will also increase (Crouse et al.,1983, Mol. Cell. Biol. 3:257).

The host cell can be co-transfected with two expression vectors, thefirst vector encoding a heavy chain derived polypeptide and the secondvector encoding a light chain derived polypeptide. The two vectors cancontain identical selectable markers that enable equal expression ofheavy and light chain polypeptides. Alternatively, a single vector canbe used to encode both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The codingsequences for the heavy and light chains can comprise cDNA or genomicDNA.

Once the antibody has been recombinantly expressed, it can be purifiedusing any method known in the art for purification of an antibody, forexample, by chromatography (e.g., ion exchange, affinity, particularlyby affinity for the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.

In yet another exemplary embodiment, the antibody is a monoclonalantibody. In any case, the hybrid antibodies have a dual specificity,preferably with one or more binding sites specific for the hapten ofchoice or one or more binding sites specific for a target antigen, forexample, an antigen associated with a tumor, an autoimmune disease, aninfectious organism, or other disease state.

Production of Antibodies

The production of antibodies will be illustrated with reference toanti-CD30 antibodies but it will be apparent for those skilled in theart that antibodies to other members of the TNF receptor family can beproduced and modified in a similar manner. The use of CD30 for theproduction of antibodies is exemplary only and not intended to belimiting.

The CD30 antigen to be used for production of antibodies may be, e.g., asoluble form of the extracellular domain of CD30 or a portion thereof,containing the desired epitope. Alternatively, cells expressing CD30 attheir cell surface (e.g., L540 (Hodgkin's lymphoma derived cell linewith a T cell phenotype) and L428 (Hodgkin's lymphoma derived cell linewith a B cell phenotype)) can be used to generate antibodies. Otherforms of CD30 useful for generating antibodies will be apparent to thoseskilled in the art.

In another exemplary embodiment, the ErbB2 antigen to be used forproduction of antibodies may be, e.g., a soluble form of theextracellular domain of ErbB2 or a portion thereof, containing thedesired epitope. Alternatively, cells expressing ErbB2 at their cellsurface (e.g., NIH-3T3 cells transformed to overexpress ErbB2; or acarcinoma cell line such as SK-BR-3 cells, see Stancovski et al. Proc.Natl. Acad. Sci. USA 88:8691-8695 (1991)) can be used to generateantibodies. Other forms of ErbB2 useful for generating antibodies willbe apparent to those skilled in the art.

(i) Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally-occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized as hereinabove described to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immmunol., 133:3001 (1984); and Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by the Scatchard analysis ofMunson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional antibody purification procedures such as, for example,protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, ormyeloma cells that do not otherwise produce antibody protein, to obtainthe synthesis of monoclonal antibodies in the recombinant host cells.Review articles on recombinant expression in bacteria of DNA encodingthe antibody include Skerra et al., Curr. Opinion in Immunol., 5:256-262(1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, monoclonal antibodies or antibody fragments canbe isolated from antibody phage libraries generated using the techniquesdescribed in McCafferty et al., Nature, 348:552-554 (1990). Clackson etal., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,222:581-597 (1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology, 10:779-783 (1992)), aswell as combinatorial infection and in vivo recombination as a strategyfor constructing very large phage libraries (Waterhouse et al., Nuc.Acids. Res., 21:2265-2266 (1993)). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy chain and light chain constant domains in placeof the homologous murine sequences (U.S. Pat. No. 4,816,567; andMorrison, et al. (1984) Proc. Natl. Acad. Sci. USA 81:6851), or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

(iii) Humanized Antibodies

A humanized antibody may have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);Verhoeyen et al., Science 239:1534-1536 (1988)), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993)).

In another embodiment, the antibodies may be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.Humanized antibodies may be prepared by a process of analysis of theparental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Various forms of the humanized antibody are contemplated. For example,the humanized antibody may be an antibody fragment, such as a Fab.Alternatively, the humanized antibody may be an intact antibody, such asan intact IgG1 antibody.

The Examples describe production of an exemplary humanized anti-ErbB2antibody. The humanized antibody may, for example, comprise nonhumanhypervariable region residues incorporated into a human variable heavydomain and may further comprise a framework region (FR) substitution ata position selected from the group consisting of 69H, 71H and 73Hutilizing the variable domain numbering system set forth in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991). Inone embodiment, the humanized antibody comprises FR substitutions at twoor all of positions 69H, 71H and 73H. Another Example describespreparation of purified trastuzumab antibody from the HERCEPTIN®formulation.

(iv) Human Antibodies

As an alternative to humanization, human antibodies can be generated.For example, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993); and U.S. Pat. Nos. 5,591,669,5,589,369 and 5,545,807.

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905. Asdiscussed above, human antibodies may also be generated by in vitroactivated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275). Humananti-CD30 antibodies are described in U.S. patent application Ser. No.10/338,366.

(v) Antibody Fragments

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Theantibody fragment may also be a “linear antibody”, e.g., as described inU.S. Pat. No. 5,641,870 for example. Such linear antibody fragments maybe monospecific or bispecific.

(vi) Bispecific Antibodies

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the CD30 protein. Alternatively, ananti-CD30 arm may be combined with an arm which binds to a Fc receptorsfor IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16)so as to focus cellular defense mechanisms to the CD30-expressing cell.Bispecific antibodies may also be used to localize cytotoxic agents tocells which express CD30.

Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991). According to a different approach, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. The fusion preferably is with an immunoglobulin heavychain constant domain, comprising at least part of the hinge, CH2, andCH3 regions. It is preferred to have the first heavy-chain constantregion (CH1) containing the site necessary for light chain binding,present in at least one of the fusions. DNAs encoding the immunoglobulinheavy chain fusions and, if desired, the immunoglobulin light chain, areinserted into separate expression vectors, and are co-transfected into asuitable host organism. This provides for great flexibility in adjustingthe mutual proportions of the three polypeptide fragments in embodimentswhen unequal ratios of the three polypeptide chains used in theconstruction provide the optimum yields. It is, however, possible toinsert the coding sequences for two or all three polypeptide chains inone expression vector when the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g., tyrosineor tryptophan). Compensatory “cavities” of identical or similar size tothe large side chain(s) are created on the interface of the secondantibody molecule by replacing large amino acid side chains with smallerones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)₂molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al., J. Immunol., 152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60(1991).

(vii) Other Amino Acid Sequence Modifications

Amino acid sequence modification(s) of the antibodies described hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the antibodies are prepared byintroducing appropriate nucleotide changes into the antibody nucleicacid, or by peptide synthesis. Such modifications include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the antibody. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. The amino acid changes also may alterpost-translational processes of the antibody, such as changing thenumber or position of glycosylation sites.

A useful method for identification of certain residues or regions of theantibody that are favored locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g., for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated.

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain.Naturally-occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent antibody from which theyare generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The antibodyvariants thus generated are displayed in a monovalent fashion fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g., binding affinity) asherein disclosed. In order to identify candidate hypervariable regionsites for modification, alanine scanning mutagenesis can be performed toidentify hypervariable region residues contributing significantly toantigen binding. Alternatively, or additionally, it may be beneficial toanalyze a crystal structure of the antigen-antibody complex to identifycontact points between the antibody and the antigen. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such variants aregenerated, the panel of variants is subjected to screening as describedherein and antibodies with superior properties in one or more relevantassays may be selected for further development.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g., so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This may be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody.Alternatively or additionally, cysteine residue(s) may be introduced inthe Fc region, thereby allowing interchain disulfide bond formation inthis region. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al. J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumoractivity may also be prepared using heterobifunctional cross-linkers asdescribed in Wolff et al. Cancer Research 53:2560-2565 (1993).Alternatively, an antibody can be engineered which has dual Fc regionsand may thereby have enhanced complement lysis and ADCC capabilities.See Stevenson et al. Anti-Cancer Drug Design 3:219-230 (1989).

To increase the serum half life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

(viii) Glycosylation Variants

Antibodies in the ADC of the invention may be glycosylated at conservedpositions in their constant regions (Jefferis and Lund, (1997) Chem.Immunol. 65:111-128; Wright and Morrison, (1997) TibTECH 15:26-32). Theoligosaccharide side chains of the immunoglobulins affect the protein'sfunction (Boyd et al., (1996) Mol. Immunol. 32:1311-1318; Wittwe andHoward, (1990) Biochem. 29:4175-4180), and the intramolecularinteraction between portions of the glycoprotein which can affect theconformation and presented three-dimensional surface of the glycoprotein(Hefferis and Lund, supra; Wyss and Wagner, (1996) Current Opin.Biotech. 7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. For example, it has been reported that in agalactosylatedIgG, the oligosaccharide moiety ‘flips’ out of the inter-CH2 space andterminal N-acetylglucosamine residues become available to bind mannosebinding protein (Malhotra et al., (1995) Nature Med. 1:237-243). Removalby glycopeptidase of the oligosaccharides from CAMPATH-1H (a recombinanthumanized murine monoclonal IgG1 antibody which recognizes the CDw52antigen of human lymphocytes) produced in Chinese Hamster Ovary (CHO)cells resulted in a complete reduction in complement mediated lysis(CMCL) (Boyd et al., (1996) Mol. Immunol. 32:1311-1318), while selectiveremoval of sialic acid residues using neuraminidase resulted in no lossof DMCL. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al. (1999) MatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Glycosylation variants of antibodies are variants in which theglycosylation pattern of an antibody is altered. By altering is meantdeleting one or more carbohydrate moieties found in the antibody, addingone or more carbohydrate moieties to the antibody, changing thecomposition of glycosylation (glycosylation pattern), the extent ofglycosylation, etc.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).Similarly, removal of glycosylation sites can be accomplished by aminoacid alteration within the native glycosylation sites of the antibody.

The amino acid sequence is usually altered by altering the underlyingnucleic acid sequence. These methods include, but are not limited to,isolation from a natural source (in the case of naturally-occurringamino acid sequence variants) or preparation by oligonucleotide-mediated(or site-directed) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared variant or a non-variant version ofthe antibody.

The glycosylation (including glycosylation pattern) of antibodies mayalso be altered without altering the amino acid sequence or theunderlying nucleotide sequence. Glycosylation largely depends on thehost cell used to express the antibody. Since the cell type used forexpression of recombinant glycoproteins, e.g., antibodies, as potentialtherapeutics is rarely the native cell, significant variations in theglycosylation pattern of the antibodies can be expected. See, e.g., Hseet al., (1997) J. Biol. Chem. 272:9062-9070. In addition to the choiceof host cells, factors which affect glycosylation during recombinantproduction of antibodies include growth mode, media formulation, culturedensity, oxygenation, pH, purification schemes and the like. Variousmethods have been proposed to alter the glycosylation pattern achievedin a particular host organism including introducing or overexpressingcertain enzymes involved in oligosaccharide production (U.S. Pat. Nos.5,047,335; 5,510,261; 5,278,299). Glycosylation, or certain types ofglycosylation, can be enzymatically removed from the glycoprotein, forexample using endoglycosidase H (Endo H). In addition, the recombinanthost cell can be genetically engineered, e.g., make defective inprocessing certain types of polysaccharides. These and similartechniques are well known in the art.

The glycosylation structure of antibodies can be readily analyzed byconventional techniques of carbohydrate analysis, including lectinchromatography, NMR, Mass spectrometry, HPLC, GPC, monosaccharidecompositional analysis, sequential enzymatic digestion, and HPAEC-PAD,which uses high pH anion exchange chromatography to separateoligosaccharides based on charge. Methods for releasing oligosaccharidesfor analytical purposes are also known, and include, without limitation,enzymatic treatment (commonly performed using peptide-N-glycosidaseF/endo-β-galactosidase), elimination using harsh alkaline environment torelease mainly O-linked structures, and chemical methods using anhydroushydrazine to release both N- and O-linked oligosaccharides.

Screening for Antibody-Drug Conjugates (ADC)

Transgenic animals and cell lines are particularly useful in screeningantibody drug conjugates (ADC) that have potential as prophylactic ortherapeutic treatments of diseases or disorders involving overexpressionof proteins including Lewis Y, CD30, CD40, and CD70. Transgenic animalsand cell lines are particularly useful in screening antibody drugconjugates (ADC) that have potential as prophylactic or therapeutictreatments of diseases or disorders involving overexpression of HER2(U.S. Pat. No. 6,632,979). Screening for a useful ADC may involveadministering candidate ADC over a range of doses to the transgenicanimal, and assaying at various time points for the effect(s) of the ADCon the disease or disorder being evaluated. Alternatively, oradditionally, the drug can be administered prior to or simultaneouslywith exposure to an inducer of the disease, if applicable. Candidate ADCmay be screened serially and individually, or in parallel under mediumor high-throughput screening format. The rate at which ADC may bescrrened for utility for prophylactic or therapeutic treatments ofdiseases or disorders is limited only by the rate of synthesis orscreening methodology, including detecting/measuring/analysis of data.

One embodiment is a screening method comprising (a) transplanting cellsfrom a stable renal cell cancer cell line into a non-human animal, (b)administering an ADC drug candidate to the non-human animal and (c)determining the ability of the candidate to inhibit the formation oftumors from the transplanted cell line.

Another embodiment is a screening method comprising (a) contacting cellsfrom a stable Hodgkin's disease cell line with an ADC drug candidate and(b) evaluating the ability of the ADC candidate to block ligandactivation of CD40.

Another embodiment is a screening method comprising (a) contacting cellsfrom a stable Hodgkin's disease cell line with an ADC drug candidate and(b) evaluating the ability of the ADC candidate to induce cell death. Inone embodiment the ability of the ADC candidate to induce apoptosis isevaluated.

One embodiment is a screening method comprising (a) transplanting cellsfrom a stable cancer cell line into a non-human animal, (b)administering an ADC drug candidate to the non-human animal and (c)determining the ability of the candidate to inhibit the formation oftumors from the transplanted cell line. The invention also concerns amethod of screening ADC candidates for the treatment of a disease ordisorder characterized by the overexpression of HER2 comprising (a)contacting cells from a stable breast cancer cell line with a drugcandidate and (b) evaluating the ability of the ADC candidate to inhibitthe growth of the stable cell line.

Another embodiment is a screening method comprising (a) contacting cellsfrom a stable cancer cell line with an ADC drug candidate and (b)evaluating the ability of the ADC candidate to block ligand activationof HER2. In one embodiment the ability of the ADC candidate to blockheregulin binding is evaluated. In another embodiment the ability of theADC candidate to block ligand-stimulated tyrosine phosphorylation isevaluated.

Another embodiment is a screening method comprising (a) contacting cellsfrom a stable cancer cell line with an ADC drug candidate and (b)evaluating the ability of the ADC candidate to induce cell death. In oneembodiment the ability of the ADC candidate to induce apoptosis isevaluated.

Another embodiment is a screening method comprising (a) administering anADC drug candidate to a transgenic non-human mammal that overexpressesin its mammary gland cells a native human HER2 protein or a fragmentthereof, wherein such transgenic mammal has stably integrated into itsgenome a nucleic acid sequence encoding a native human HER2 protein or afragment thereof having the biological activity of native human HER2,operably linked to transcriptional regulatory sequences directing itsexpression to the mammary gland, and develops a mammary tumor notresponding or poorly responding to anti-HER2 antibody treatment, or to anon-human mammal bearing a tumor transplanted from said transgenicnon-human mammal; and (b) evaluating the effect of the ADC candidate onthe target disease or disorder. Without limitations, the disease ordisorder may be a HER2-overexpressing cancer, such as breast, ovarian,stomach, endometrial, salivary gland, lung, kidney, colon, thyroid,pancreatic and bladder cancer. The cancer preferably is breast cancerwhich expressed HER2 in at least about 500,000 copies per cell, morepreferably at least about 2,000,000 copies per cell. ADC drug candidatesmay, for example, be evaluated for their ability to induce cell deathand/or apoptosis, using assay methods well known in the art anddescribed hereinafter.

In one embodiment, candidate ADC are screened by being administered tothe transgenic animal over a range of doses, and evaluating the animal'sphysiological response to the compounds over time. Administration may beoral, or by suitable injection, depending on the chemical nature of thecompound being evaluated. In some cases, it may be appropriate toadminister the compound in conjunction with co-factors that wouldenhance the efficacy of the compound. If cell lines derived from thesubject transgenic animals are used to screen for compounds useful intreating various disorders, the test compounds are added to the cellculture medium at an appropriate time, and the cellular response to thecompound is evaluated over time using the appropriate biochemical and/orhistological assays. In some cases, it may be appropriate to apply thecompound of interest to the culture medium in conjunction withco-factors that would enhance the efficacy of the compound.

Thus, provided herein are assays for identifying ADC which specificallytarget and bind a target protein, the presence of which is correlatedwith abnormal cellular function, and in the pathogenesis of cellularproliferation and/or differentiation that is causally related to thedevelopment of tumors.

To identify an ADC which blocks ligand activation of an ErbB (e.g.,ErbB2) receptor, the ability of the compound to block ErbB ligandbinding to cells expressing the ErbB (ErbB2) receptor (e.g., inconjugation with another ErbB receptor with which the ErbB receptor ofinterest forms an ErbB hetero-oligomer) may be determined. For example,cells isolated from the transgenic animal overexpressing HER2 andtransfected to express another ErbB receptor (with which HER2 formshetero-oligomer) may be incubated, i.e. culturing, with the ADC and thenexposed to labeled ErbB ligand. The ability of the compound to blockligand binding to the ErbB receptor in the ErbB hetero-oligomer may thenbe evaluated.

For example, inhibition of heregulin (HRG) binding to breast tumor celllines, overexpressing HER2 and established from the transgenic non-humanmammals (e.g., mice) herein, by the candidate ADC may be performed usingmonolayer cultures on ice in a 24-well-plate format. Anti-ErbB2monoclonal antibodies may be added to each well and incubated for 30minutes. ¹²⁵I-labeled rHMGβ1₁₇₇₋₂₂₄ (25,000 cpm) may then be added, andthe incubation may be continued for 4 to 16 hours. Dose response curvesmay be prepared and an IC₅₀ value (cytotoxic activity) may be calculatedfor the compound of interest.

Alternatively, or additionally, the ability of an ADC to block ErbBligand-stimulated tyrosine phosphorylation of an ErbB receptor presentin an ErbB hetero-oligomer may be assessed. For example, cell linesestablished from the transgenic animals herein may be incubated with atest ADC and then assayed for ErbB ligand-dependent tyrosinephosphorylation activity using an anti-phosphotyrosine monoclonalantibody (which is optionally conjugated with a detectable label). Thekinase receptor activation assay described in U.S. Pat. No. 5,766,863 isalso available for determining ErbB receptor activation and blocking ofthat activity by the compound.

In one embodiment, one may screen for ADC which inhibit HRG stimulationof p180 tyrosine phosphorylation in MCF7 cells essentially as describedbelow. For example, a cell line established from a HER2-transgenicanimal may be plated in 24-well plates and the compound may be added toeach well and incubated for 30 minutes at room temperature; thenrHRGβ₁₇₇₋₂₄₄ may be added to each well to a final concentration of 0.2nM, and the incubation may be continued for about 8 minutes. Media maybe aspirated from each well, and reactions may be stopped by theaddition of 100 μl of SDS sample buffer (5% SDS, 25 mM DTT, and 25 mMTris-HCl, pH 6.8). Each sample (25 μl) may be electrophoresed on a 4-12%gradient gel (Novex) and then electrophoretically transferred topolyvinylidene difluoride membrane. Antiphosphotyrosine (at 1 μg/ml)immunoblots may be developed, and the intensity of the predominantreactive band at M_(r)-180,000 may be quantified by reflectancedensitometry. An alternate method to evaluate inhibition of receptorphosphorylation is the KIRA (kinase receptor activation) assay of Sadicket al. (1998) Jour. of Pharm. and Biomed. Anal. Some of the wellestablished monoclonal antibodies against HER2 that are known to inhibitHRG stimulation of p180 tyrosine phosphorylation can be used as positivecontrol in this assay. A dose-response curve for inhibition of HRGstimulation of p180 tyrosine phosphorylation as determined byreflectance densitometry may be prepared and an IC₅₀ for the compound ofinterest may be calculated.

One may also assess the growth inhibitory effects of a test ADC on celllines derived from a HER2-transgenic animal, e.g., essentially asdescribed in Schaefer et al. (1997) Oncogene 15:1385-1394. According tothis assay, the cells may be treated with a test compound at variousconcentrations for 4 days and stained with crystal violet or the redoxdye Alamar Blue. Incubation with the compound may show a growthinhibitory effect on this cell line similar to that displayed bymonoclonal antibody 2C4 on MDA-MB-175 cells (Schaefer et al., supra). Ina further embodiment, exogenous HRG will not significantly reverse thisinhibition.

To identify growth inhibitory compounds that specifically target anantigen of interest, one may screen for compounds which inhibit thegrowth of cancer cells overexpressing antigen of interest derived fromtransgenic animals, the assay described in U.S. Pat. No. 5,677,171 canbe performed. According to this assay, cancer cells overexpressing theantigen of interst are grown in a 1:1 mixture of F12 and DMEM mediumsupplemented with 10% fetal bovine serum, glutamine and penicillinstreptomycin. The cells are plated at 20,000 cells in a 35 mm cellculture dish (2 mls/35 mm dish) and the test compound is added atvarious concentrations. After six days, the number of cells, compared tountreated cells is counted using an electronic COULTER™ cell counter.Those compounds which inhibit cell growth by about 20-100% or about50-100% may be selected as growth inhibitory compounds.

To select for compounds which induce cell death, loss of membraneintegrity as indicated by, e.g., PI, trypan blue or 7AAD uptake may beassessed relative to control. The PI uptake assay uses cells isolatedfrom the tumor tissue of interest of a transgenic animal. According tothis assay, the cells are cultured in Dulbecco's Modified Eagle Medium(D-MEM):Ham's F-12 (50:50) supplemented with 10% heat-inactivated FBS(Hyclone) and 2 mM L-glutamine. Thus, the assay is performed in theabsence of complement and immune effector cells. The cells are seeded ata density of 3×10⁶ per dish in 100×20 mm dishes and allowed to attachovernight. The medium is then removed and replaced with fresh mediumalone or medium containing various concentrations of the compound. Thecells are incubated for a 3-day time period. Following each treatment,monolayers are washed with PBS and detached by trypsinization. Cells arethen centrifuged at 1200 rpm for 5 minutes at 4° C., the pelletresuspended in 3 ml cold Ca²⁺ binding buffer (10 mM Hepes, pH 7.4, 140mM NaCl, 2.5 mM CaCl₂) and aliquoted into 35 mm strainer-capped 12×75 mmtubes (1 ml per tube, 3 tubes per treatment group) for removal of cellclumps. Tubes then receive PI (10 μg/ml). Samples may be analyzed usinga FACSCAN™ flow cytometer and FACSCONVERTTM CellQuest software (BectonDickinson). Those compounds which induce statistically significantlevels of cell death as determined by PI uptake may be selected as celldeath-inducing compounds.

In order to select for compounds which induce apoptosis, an annexinbinding assay using cells established from the tumor tissue of interestof the transgenic animal is performed. The cells are cultured and seededin dishes as discussed in the preceding paragraph. The medium is thenremoved and replaced with fresh medium alone or medium containing 10μg/ml of the antibody drug conjugate (ADC). Following a three-dayincubation period, monolayers are washed with PBS and detached bytrypsinization. Cells are then centrifuged, resuspended in Ca²⁺ bindingbuffer and aliquoted into tubes as discussed above for the cell deathassay. Tubes then receive labeled annexin (e.g., annexin V-FITC) (1μg/ml). Samples may be analyzed using a FACSCAN™ flow cytometer andFACSCONVERT™ CellQuest software (Becton Dickinson). Those compoundswhich induce statistically significant levels of annexin bindingrelative to control are selected as apoptosis-inducing compounds.

In Vitro Cell Proliferation Assays

Generally, the cytotoxic or cytostatic activity of an antibody drugconjugate (ADC) is measured by: exposing mammalian cells having receptorproteins to the antibody of the ADC in a cell culture medium; culturingthe cells for a period from about 6 hours to about 5 days; and measuringcell viability. Cell-based in vitro assays were used to measureviability (proliferation), cytotoxicity, and induction of apoptosis(caspase activation) of the ADC of the invention.

The in vitro potency of antibody drug conjugates was measured by a cellproliferation assay (Example 18, FIGS. 7-10). The CellTiter-Glo®Luminescent Cell Viability Assay is a commercially available (PromegaCorp., Madison, Wis.), homogeneous assay method based on the recombinantexpression of Coleoptera luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713and 5700670). This cell proliferation assay determines the number ofviable cells in culture based on quantitation of the ATP present, anindicator of metabolically active cells (Crouch et al. (1993) J.Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677). The CellTiter-Glo®Assay was conducted in 96 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al. (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing. The cells may be treated continuously with ADC, or they may betreated and separated from ADC. Generally, cells treated briefly, i.e. 3hours, showed the same potency effects as continuously treated cells.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used (FIG. 24). Viable cells are reflected inrelative luminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g., 96 or 384 well format. Data can be recorded byluminometer or CCD camera imaging device. The luminescence output ispresented as relative light units (RLU), measured over time.

The anti-proliferative effects of antibody drug conjugates were measuredby the cell proliferation, in vitro cell killing assay above againstfour different breast tumor cell lines (FIGS. 7-10). IC₅₀ values wereestablished for SK-BR-3 and BT-474 which are known to over express HER2receptor protein. Table 2a shows the potency (IC₅₀) measurements ofexemplary antibody drug conjugates in the cell proliferation assayagainst SK-BR-3 cells. Table 2b shows the potency (IC₅₀) measurements ofexemplary antibody drug conjugates in the cell proliferation assayagainst BT-474 cells.

Antibody drug conjugates: Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab;Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab; Trastuzumab-MC-MMAF, 4.1MMAF/Ab; Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab;Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab; and Trastuzumab-MC-vc-PAB-MMAF,3.7 MMAF/Ab did not inhibit the proliferation of MCF-7 cells (FIG. 9).

Antibody drug conjugates: Trastuzumab-MC-vc-PAB-MMAE, 4.1 MMAE/Ab;Trastuzumab-MC-vc-PAB-MMAE, 3.3 MMAE/Ab; Trastuzumab-MC-vc-PAB-MMAF, 3.7MMAF/Ab; Trastuzumab-MC-vc-PAB-MMAF, 3.8 MMAF/Ab;Trastuzumab-MC-(N-Me)vc-PAB-MMAF, 3.9 MMAF/Ab; and Trastuzumab-MC-MMAF,4.1 MMAF/Ab did not inhibit the proliferation of MDA-MB-468 cells (FIG.10).

MCF-7 and MDA-MB-468 cells do not overexpress HER2 receptor protein. Theanti-HER2 antibody drug conjugates of the invention therefore showselectivity for inhibition of cells which express HER2.

TABLE 2a SK-BR-3 cells Antibody Drug Conjugate H = trastuzumab linkedvia a cysteine [cys] IC₅₀ except where noted (μg ADC/ml) H-MC-MMAF, 4.1MMAF/Ab 0.008 H-MC-MMAF, 4.8 MMAF/Ab 0.002 H-MC-vc-PAB-MMAE, 0.007H-MC-vc-PAB-MMAE 0.015 H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab 0.0035-0.01H-MC-vc-PAB-MMAF, 4.4 MMAF/Ab 0.006-0.007 H-MC-vc-PAB-MMAF, 4.8 MMAF/Ab0.006 H-MC-(N—Me)vc-PAB-MMAF, 3.9 MMAF/Ab 0.0035 H-MC-MMAF, 4.1 MMAF/Ab0.0035 H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab 0.010 H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab0.007 H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab 0.015 H-MC-vc-PAB-MMAF, 3.7 MMAF/Ab.0.010 H-MC-vc-PAB-MMAE, 7.5 MMAE/Ab 0.0025 H-MC-MMAE, 8.8 MMAE/Ab 0.018H-MC-MMAE, 4.6 MMAE/Ab 0.05 H-MC-(L)val-(L)cit-PAB-MMAE, 8.7 0.0003MMAE/Ab H-MC-(D)val-(D)cit-PAB-MMAE, 8.2 0.02 MMAE/AbH-MC-(D)val-(L)cit-PAB-MMAE, 8.4 0.0015 MMAE/AbH-MC-(D)val-(L)cit-PAB-MMAE, 3.2 0.003 MMAE/Ab H-Trastuzumab 0.083H-vc-MMAE, linked via a lysine [lys] 0.002 H-phe-lys-MMAE, linked via alysine [lys] 0.0015 4D5-Fc8-MC-vc-PAB-MMAF, 4.4 MMAF/Ab 0.004Hg-MC-vc-PAB-MMAF, 4.1 MMAF/Ab 0.01 7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab 0.014D5 Fab-MC-vc-PAB-MMAF, 1.5 MMAF/Ab 0.02 Anti-TF Fab-MC-vc-PAB-MMAE* —

TABLE 2b BT474 cells Antibody Drug Conjugate IC₅₀ H = trastuzumab linkedvia a cysteine [cys] (μg ADC/ml) H-MC-MMAF, 4.1 MMAF/Ab 0.008 H-MC-MMAF,4.8 MMAF/Ab 0.002 H-MC-vc-PAB-MMAE, 4.1 MMAE/Ab 0.015 H-MC-vc-PAB-MMAF,3.8 MMAF/Ab 0.02-0.05 H-MC-vc-PAB-MMAF, 4.4 MMAF/Ab 0.01H-MC-vc-PAB-MMAF, 4.8 MMAF/Ab 0.01 H-MC-vc-PAB-MMAE, 3.3 MMAE/Ab 0.02H-MC-vc-PAB-MMAF, 3.7 MMAF/Ab. 0.02 H-MC-vc-PAB-MMAF, 3.8 MMAF/Ab 0.015H-MC-(N—Me)vc-PAB-MMAF, 3.9 MMAF/Ab 0.010 H-MC-MMAF, 4.1 MMAF/Ab 0.00015H-MC-vc-PAB-MMAE, 7.5 MMAE/Ab 0.0025 H-MC-MMAE, 8.8 MMAE/Ab 0.04H-MC-MMAE, 4.6 MMAE/Ab 0.07 4D5-Fc8-MC-vc-PAB-MMAF, 4.4 MMAF/Ab 0.008Hg-MC-vc-PAB-MMAF, 4.1 MMAF/Ab 0.01 7C2-MC-vc-PAB-MMAF, 4.0 MMAF/Ab0.015 4D5 Fab-MC-vc-PAB-MMAF, 1.5 MMAF/Ab 0.04 Anti-TFFab-MC-vc-PAB-MMAE* — H = trastuzumab 7C2 = anti-HER2 murine antibodywhich binds a different epitope than trastuzumab. Fc8 = mutant that doesnot bind to FcRn Hg = “Hingeless” full-length humanized 4D5, with heavychain hinge cysteines mutated to serines. Expressed in E. coli(therefore non-glycosylated.) Anti-TF Fab = anti-tissue factor antibodyfragment *activity against MDA-MB-468 cells

In a surprising and unexpected discovery, the in vitro cellproliferation activity results of the ADC in Tables 2a and 2b showgenerally that ADC with a low average number of drug moieties perantibody showed efficacy, e.g., IC₅₀<0.1 μg ADC/ml. The results suggestthat at least for trastuzumab ADC, the optimal ratio of drug moietiesper antibody may be less than 8, and may be about 2 to about 5.

In Vivo Plasma Clearance and Stability

Pharmacokinetic plasma clearance and stability of ADC were investigatedin rats and cynomolgus monkeys. Plasma concentration was measured overtime. Table 2c shows pharmacokinetic data of antibody drug conjugatesand other dosed samples in rats. Rats are a non-specific model for ErbBreceptor antibodies, since the rat is not known to express HER2 receptorproteins.

TABLE 2c Pharmacokinetics in Rats CL AUCinf mL/ Cmax T ½ Sample day*day/ μg/ Term. % dose mg/kg μg/mL kg mL days Conj. H-MC-vc-PAB-MMAE(Total 78.6 26.3 39.5 5.80 40.6 Ab H-MC-vc-PAB-MMAE 31.1 64.4 33.2 3.00(Conj.) H-MC-vc-PAB-MMAF (Total 170 12.0 47.9 8.4 50.0 Ab)H-MC-vc-PAB-MMAF 83.9 24.0 44.7 4.01 (Conj.) H-MC-MMAE (Total Ab) 27918.9 79.6 7.65 33 H-MC-MMAE (Conj.) 90.6 62.9 62.9 4.46 5 mg/kgH-MC-MMAF (Total Ab) 299 6.74 49.1 11.6 37 H-MC-MMAF (Conj.) 110 18.2650.2 4.54 H-MC-vc-MMAF, wo/PAB, 306 6.6 78.7 11.9 19.6 (Total Ab)H-MC-vc-MMAF, wo/PAB, 59.9 33.4 82.8 2.1 (Conj.) H-Me-vc-PAB-MMAF (Total186 10.8 46.9 8.3 45.3 Ab) H-Me-vc-PAB-MMAF 84.0 23.8 49.6 4.3 (Conj.)H-Me-vc-PAB-MMAE (Total 135 15.0 44.9 11.2 23.8 Ab) H-Me-vc-PAB-MMAE31.9 63.8 45.2 3.0 (Conj.) H-MC-vc-MMAF, wo/PAB, 306 6.6 78.7 11.9 19.6(Total Ab) H-MC-vc-MMAF, wo/PAB, 59.9 33.4 82.8 2.1 (Conj.)H-MC-(D)val-(L)cit-PAB- 107 19.2 30.6 9.6 38.1 MMAE (Total Ab)H-MC-(D)val-(L)cit-PAB- 40 50.4 33.7 3.98 MMAE (Conj.)H-MC-(Me)-vc-PAB-MMAE, 135.1 15.0 44.9 11.2 23.8 Total AbH-MC-(Me)-vc-PAB-MMAE, 31.9 63.8 45.2 2.96 Conj. H-MC-(D)val-(D)cit-PAB-88.2 22.8 33.8 10.5 38.3 MMAE, Total Ab H-MC-(D)val-(D)cit-PAB- 33.659.8 36.0 4.43 MMAE, Conj. H-MC-vc-PAB-MMAE, Total 78.6 26.3 39.5 5.840.6 Ab H-MC-vc-PAB-MMAE, Conj. 31.1 64.4 33.2 3.00 H linked to MC bylysine [lys] MMAF 0.99 204 280 0.224 — 200 μg/kg MMAE 3.71 62.6 6490.743 — 206 μg/kg HER F(ab′)₂-MC-vc-MMAE, 9.3 217 34.4 0.35 95 Total AbHER F(ab′)₂-MC-vc-MMAE, 8.8 227 36.9 0.29 Conj. 4D5-H-Fab-MC-vc-MMAF,43.8 46.2 38.5 1.49 68 Total Ab 4D5-H-Fab-MC-vc-MMAF, 29.9 68.1 34.11.12 Conj. 4D5-H-Fab-MC-vc-MMAE, 71.5 70.3 108 1.18 59 Total Ab4D5-H-Fab-MC-vc-MMAE, 42.2 118.9 114 0.74 Conj. 4D5-H-Fab 93.4 53.9 1331.08 — H-MC-vc-PAB-MMAF, Total 170 12.03 47.9 8.44 49.5 AbH-MC-vc-PAB-MMAF, Conj. 83.9 23.96 44.7 4.01 H-MC-vc-PAB-MMAF- 211 9.839.8 8.53 34.3 DMAEA, Total Ab H-MC-vc-PAB-MMAF- 71.5 28.2 38.8 3.64DMAEA, Conj. H-MC-vc-PAB-MMAF-TEG, 209 9.75 53.2 8.32 29.7 Total AbH-MC-vc-PAB-MMAF-TEG, 63.4 31.8 34.9 4.36 Conj. H = trastuzumab linkedvia a cysteine [cys] except where noted 2 mg/kg dose except where noted

AUC inf is the area under the plasma concentration-time curve from timeof dosing to infinity and is a measure of the total exposure to themeasured entity (drug, ADC). CL is defined as the volume of plasmacleared of the measured entity in unit time and is expressed bynormalizing to body weight. T1/2 term is the half-life of the drug inthe body measured during its elimination phase. The % Conj. term is therelative amount of ADC compared to total antibody detected, by separateELISA immunoaffinity tests (“Analytical Methods for BiotechnologyProducts”, Ferraiolo et al, p85-98 in Pharmacokinetics of Drugs (1994)P. G. Welling and L. P. Balant, Eds., Handbook of ExperimentalPharmacology, Vol. 110, Springer-Verlag. The % Conj. calculation issimply AUCinf of ADC÷AUCinf total Ab, and is a general indicator oflinker stability, although other factors and mechanisms may be ineffect.

FIG. 11 shows a graph of a plasma concentration clearance study afteradministration of the antibody drug conjugates: H-MC-vc-PAB-MMAF-TEG andH-MC-vc-PAB-MMAF to Sprague-Dawley rats. Concentrations of totalantibody and ADC were measured over time.

FIG. 12 shows a graph of a two stage plasma concentration clearancestudy where ADC was administered at different dosages and concentrationsof total antibody and ADC were measured over time.

In Vivo Efficacy

The in vivo efficacy of the ADC of the invention was measured by a highexpressing HER2 transgenic explant mouse model. An allograft waspropagated from the Fo5 mmtv transgenic mouse which does not respond to,or responds poorly to, HERCEPTIN® therapy. Subjects were treated oncewith ADC and monitored over 3-6 weeks to measure the time to tumordoubling, log cell kill, and tumor shrinkage. Follow up dose-responseand multi-dose experiments were conducted.

Tumors arise readily in transgenic mice that express a mutationallyactivated form of neu, the rat homolog of HER2, but the HER2 that isoverexpressed in breast cancers is not mutated and tumor formation ismuch less robust in transgenic mice that overexpress nonmutated HER2(Webster et al. (1994) Semin. Cancer Biol. 5:69-76).

To improve tumor formation with nonmutated HER2, transgenic mice wereproduced using a HER2 cDNA plasmid in which an upstream ATG was deletedin order to prevent initiation of translation at such upstream ATGcodons, which would otherwise reduce the frequency of translationinitiation from the downstream authentic initiation codon of HER2 (forexample, see Child et al. (1999) J. Biol. Chem. 274: 24335-24341).Additionally, a chimeric intron was added to the 5′ end, which shouldalso enhance the level of expression as reported earlier (Neuberger andWilliams (1988) Nucleic Acids Res. 16: 6713; Buchman and Berg (1988)Mol. Cell. Biol. 8:4395; Brinster et al. (1988) Proc. Natl. Acad. Sci.USA 85:836). The chimeric intron was derived from a Promega vector,pCI-neo mammalian expression vector (bp 890-1022). The cDNA 3′-end isflanked by human growth hormone exons 4 and 5, and polyadenylationsequences. Moreover, FVB mice were used because this strain is moresusceptible to tumor development. The promoter from MMTV-LTR was used toensure tissue-specific HER2 expression in the mammary gland. Animalswere fed the AIN 76A diet in order to increase susceptibility to tumorformation (Rao et al. (1997) Breast Cancer Res. and Treatment45:149-158).

TABLE 2d Tumor measurements in allograft mouse model - MMTV-HER2 Fo5Mammary Tumor, athymic nude mice single dose at day 1 (T = 0) exceptwhere noted H = trastuzumab linked via a cysteine [cys] except wherenoted Tumor doubling Mean Sample time log cell Drugs per antibody DoseTi PR CR (days) kill Vehicle 2-5 0 H-MC-vc- 1250 μg/m² 5/5 4/7 0/7 181.5 PAB-MMAE 8.7 MMAE/Ab H-MC-vc- 555 μg/m² 2/5 2/7 5/7 69 6.6 PAB-MMAF3.8 MMAF/Ab H-MC(Me)-vc-PAB- >50 6.4 MMAF H-MC-MMAF 9.2 mg/kg Ab 7/7 6/70/7 63 9 4.8 MMAF/Ab 550 μg/m² at 0, 7, 14 and 21 days H-MC-MMAF 14mg/kg Ab 5/5 5/7 2/7 >63 4.8 MMAF/Ab 840 μg/m² at 0, 7, 14 and 21 daysH-MC-vc- 3.5 mg/kg Ab 5/6 1/7 3/7 >36 PAB-MMAF 5.9 MMAF/Ab 300 μg/m² at0, 21, and 42 days H-MC-vc- 4.9 mg/kg Ab 4/7 2/7 5/7 >90 PAB-MMAF 5.9MMAF/Ab 425 μg/m² at 0, 21, and 42 days H-MC-vc- 6.4 mg/kg Ab 3/6 1/76/7 >90 PAB-MMAF 5.9 MMAF/Ab 550 μg/m² at 0, 21, and 42 days H-(L)val-10 mg/kg 7/7 1/7 0/7 15.2 1.1 (L)cit-MMAE 8.7 MMAE/Ab H-MC-MMAE 10 mg/kg7/7 0/7 0/7 4 0.1 4.6 MMAE/Ab H-(D)val-(D)cit- 10 mg/kg 7/7 0/7 0/7 3MMAE 4.2 MMAE/Ab H-(D)val- 13 mg/kg 7/7 0/7 0/7 9 0.6 (L)cit-MMAE 3.2MMAE/Ab H-MC(Me)- 13 mg/kg 7/7 3/7 0/7 17 1.2 vc-MMAE 3.0 MMAE/AbH-(L)val- 12 mg/kg 7/7 0/7 0/7 5 0.2 (D)cit-MMAE 3.5 MMAE/Ab H-vc-MMAE10 mg/kg 7/7 17 8.7 MMAE/Ab H-cys-vc-MMAF 1 mg/kg 7/7 3 3.8 MMAF/AbH-cys-vc-MMAF 3 mg/kg 7/7 >17 3.8 MMAF/Ab H-cys-vc-MMAF 10 mg/kg 4/7 4/73/7 >17 3.8 MMAF/Ab H-MC-vc- 10 mg/kg 3/6 1/7 6/7 81 7.8 MMAF-TEG 4MMAF/Ab H-MC-vc- 10 mg/kg 0/5 0/7 7/7 81 7.9 MMAF-TEG 4 MMAF/Ab q3 wk ×3 H-vc-MMAF (lot 1) 10 mg/kg 4/6 2/8 5/8 H-vc-MMAF (lot 2) 10 mg/kg 7/81/8 1/8 H-MC-MMAF 10 mg/kg 8/8 1/8 0/8 18 550 μg/m² H-(Me)-vc-MMAF 10mg/kg 3/7 2/8 5/8 H-vc-MMAE 3.7 mg/kg 6/6 0/7 1/7 17 2.3 7.5 MMAE/Ab at0, 7, 14, 21, 28 days H-vc-MMAE 7.5 mg/kg 5/7 3/7 3/7 69 10 7.5 MMAE/Abat 0, 7, 14, 21, 28 days anti IL8-vc-MMAE 7.5 mg/kg 7/7 0/7 0/7 5 0.57.5 MMAE/Ab at 0, 7, 14, 21, 28 days anti IL8-vc-MMAE 3.7 mg/kg 6/6 0/70/7 3 0.2 7.5 MMAE/Ab at 0, 7, 14, 21, 28 days H-fk-MMAE 7.5 mg/kg 7/71/7 0/7 31 4.4 7.5 MMAE/Ab at 0, 7, 14, 21, 28 days H-fk-MMAE 3.7 mg/kg7/7 0/7 0/7 8.3 0.9 7.5 MMAE/Ab at 0, 7, 14, 21, 28 days antiIL8-fk-MMAE 7.5 mg/kg 7/7 0/7 0/7 6 0.5 7.5 MMAE/Ab at 0, 7, 14, 21, 28days anti IL8-fk-MMAE 3.7 mg/kg 7/7 0/7 0/7 3 0.1 7.5 MMAE/Ab at 0, 7,14, 21, 28 days Trastuzumab 7.5 mg/kg 7/7 0/7 0/7 5 0.4 at 0, 7, 14, 21,28 days H-vc-MMAE 10 mg/kg 6/6 3/6 0/6 15 1.3 8.7 MMAE/Ab 1250 μg/m²H-vc-MMAE 10 mg/kg 7/7 5/7 >19 1250 μg/m² at 0, 7, and 14 days H-vc-MMAE3 mg/kg 7/7 8 at 0, 7, and 14 days H-vc-MMAE 1 mg/kg 7/7 7 at 0, 7, and14 days H-vc-MMAF 10 mg/kg 8/8 5/8 >21 H-vc-MMAF 10 mg/kg 4/7 4/73/7 >21 at 0, 7, and 14 days H-vc-MMAF 3 mg/kg 7/7 6 at 0, 7, and 14days H-vc-MMAF 1 mg/kg 8/8 4 at 0, 7, and 14 days Trastuzumab 10 mg/kg8/8 3 at 0 and 7 days Hg-MC-vc-PAB- 10 mg/kg 6/7 3/8 5/8 56 5.1 MMAF at0 days 4.1 MMAF/Ab Fc8-MC-vc-PAB- 10 mg/kg 7/7 6/8 0/8 25 2.1 MMAF at 0days 4.4 MMAF/Ab 7C2-MC-vc-PAB- 10 mg/kg 5/6 6/8 1/8 41 3.7 MMAF at 0days 4 MMAF/Ab H-MC-vc- 10 mg/kg 3/8 3/8 5/8 62 5.7 PAB-MMAF 5.9 MMAF/Abat 0 days 2H9-MC-vc-PAB- 9/9 >14 days MMAE 2H9-MC-vc-PAB- 9/9 >14 daysMMAF 11D10-vc- 9/9 >14 days PAB-MMAE 11D10-vc- 9/9  11 days PAB-MMAF 7C2= anti-HER2 murine antibody which binds a different epitope thantrastuzumab. Fc8 = mutant that does not bind to FcRn Hg = “Hingeless”full-length humanized 4D5, with heavy chain hinge cysteines mutated toserines. Expressed in E. coli (therefore non-glycosylated.) 2H9 =Anti-EphB2R 11D10 = Anti-0772P

The term Ti is the number of animals in the study group with tumor atT=0÷total animals in group. The term PR is the number of animalsattaining partial remission of tumor÷animals with tumor at T=0 in group.The term CR is the number of animals attaining complete remission oftumor÷animals with tumor at T=0 in group. The term Log cell kill is thetime in days for the tumor volume to double—the time in days for thecontrol tumor volume to double divided by 3.32X time for tumor volume todouble in control animals (dosed with Vehicle). The log-cell-killcalculation takes into account tumor growth delay resulting fromtreatment and tumor volume doubling time of the control group.Anti-tumor activity of ADC is classified with log-cell-kill values of:

++++ ≧ 3.4 (highly active) +++ = 2.5-3.4 ++ = 1.7-2.4 + = 1.0-1.6inactive = 0

FIG. 13 shows the mean tumor volume change over time in athymic nudemice with MMTV-HER2Fo5 Mammary tumor allografts dosed on Day 0 with:Vehicle, Trastuzumab-MC-vc-PAB-MMAE (1250 μg/m²) andTrastuzumab-MC-vc-PAB-MMAF (555 μg/m²). (H=Trastuzumab). The growth oftumors was retarded by treatment with ADC as compared to control(Vehicle) level of growth. FIG. 14 shows the mean tumor volume changeover time in athymic nude mice with MMTV-HER2Fo5 Mammary tumorallografts dosed on Day 0 with 10 mg/kg (660 μg/m²) ofTrastuzumab-MC-MMAE and 1250 μg/m² Trastuzumab-MC-vc-PAB-MMAE. FIG. 15shows the mean tumor volume change over time in athymic nude mice withMMTV-HER2Fo5 Mammary tumor allografts dosed with 650 μg/m²Trastuzumab-MC-MMAF. Table 2d and FIGS. 13-15 show that the ADC havestrong anti-tumor activity in the allograft of a HER2 positive tumor(Fo5) that originally arose in an MMTV-HER2 transgenic mouse. Theantibody alone (e.g., Trastuzumab) does not have significant anti-tumoractivity in this model (Erickson et al. U.S. Pat. No. 6,632,979). Asillustrated in FIGS. 13-15, the growth of the tumors was retarded bytreatment with ADC as compared to control (Vehicle) level of growth.

In a surprising and unexpected discovery, the in vivo anti-tumoractivity results of the ADC in Table 2d show generally that ADC with alow average number of drug moieties per antibody showed efficacy, e.g.,tumor doubling time >15 days and mean log cell kill >1.0. FIG. 16 showsthat for the antibody drug conjugate, trastuzumab-MC-vc-PAB-MMAF, themean tumor volume diminished and did not progress where theMMAF:trastuzumab ratio was 2 and 4, whereas tumor progressed at a ratioof 5.9 and 6, but at a rate lower than Vehicle (buffer). The rate oftumor progression in this mouse xenograft model was about the same, i.e.3 days, for Vehicle and trastuzumab. The results suggest that at leastfor trastuzumab ADC, the optimal ratio of drug moieties per antibody maybe less than about 8, and may be about 2 to about 4.

Rodent Toxicity

Antibody drug conjugates and an ADC-minus control, “Vehicle”, wereevaluated in an acute toxicity rat model. Toxicity of ADC wasinvestigated by treatment of male and female Sprague-Dawley rats withthe ADC and subsequent inspection and analysis of the effects on variousorgans. Gross observations included changes in body weights and signs oflesions and bleeding. Clinical pathology parameters (serum chemistry andhematology), histopathology, and necropsy were conducted on dosedanimals.

It is considered that weight loss, or weight change relative to animalsdosed only with Vehicle, in animals after dosing with ADC is a gross andgeneral indicator of systemic or localized toxicity. FIGS. 17-19 showthe effects of various ADC and control (Vehicle) after dosing on ratbody weight.

Hepatotoxicity was measured by elevated liver enzymes, increased numbersof mitotic and apoptotic figures and hepatocyte necrosis. Hematolymphoidtoxicity was observed by depletion of leukocytes, primarily granuloctyes(neutrophils), and/or platelets, and lymphoid organ involvement, i.e.atrophy or apoptotic activity. Toxicity was also noted bygastrointestinal tract lesions such as increased numbers of mitotic andapoptotic figures and degenerative enterocolitis.

Enzymes indicative of liver injury that were studied include:

AST (aspartate aminotransferase)

Localization: cytoplasmic; liver, heart, skeletal muscle, kidney

Liver:Plasma ratio of 7000:1

T1/2: 17 hrs

ALT (alanine aminotransferase)

Localization: cytoplasmic; liver, kidney, heart, skeletal muscle

Liver:Plasma ratio of 3000:1

-   -   T1/2: 42 hrs; diurnal variation    -   GGT (g-glutamyl transferase)    -   Localization: plasma membrane of cells with high secretory or        absorptive capacity; liver, kidney, intestine    -   Poor predictor of liver injury; commonly elevated in bile duct        disorders

The toxicity profiles of trastuzumab-MC-val-cit-MMAF,trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF andtrastuzumab-MC-val-cit-PAB-MMAF were studied in female Sprague-Dawleyrats (Example 19). The humanized trastuzumab antibody does not bindappreciably to rat tissue, and any toxicity would be considerednon-specific. Variants at dose levels of 840 and 2105 ug/m² MMAF werecompared to trastuzumab-MC-val-cit-PAB-MMAF at 2105 ug/m².

Animals in groups 1, 2, 3, 4, 6, and 7 (Vehicle, 9.94 & 24.90 mg/kgtrastuzumab-MC-val-cit-MMAF, 10.69 mg/kgtrastuzumab-MC(Me)-val-cit-PAB-MMAF, and 10.17 & 25.50 mg/kgtrastuzumab-MC-MMAF, respectively) gained weight during the study.Animals in groups 5 and 8 (26.78 mg/kgtrastuzumab-MC(Me)-val-cit-PAB-MMAF and 21.85 mg/kgtrastuzumab-MC-val-cit-PAB-MMAF, respectively) lost weight during thestudy. On Study Day 5, the change in body weights of animals in groups2, 6 and 7 were not significantly different from group 1 animals. Thechange in body weights of animals in groups 3, 4, 5 and 8 werestatistically different from group 1 animals (Example 19).

Rats treated with trastuzumab-MC-MMAF (groups 6 and 7) wereindistinguishable from vehicle-treated control animals at both doselevels; i.e. this conjugate showed a superior safety profile in thismodel. Rats treated with trastuzumab-MC-val-cit-MMAF (without theself-immolative PAB moiety; groups 2 and 3) showed dose-dependentchanges typical for MMAF conjugates; the extent of the changes was lesscompared with a full length MC-val-cit-PAB-MMAF conjugate (group 8). Theplatelet counts on day 5 were at approximately 30% of baseline values inanimals of group 3 (high dose trastuzumab-MC-val-cit-MMAF) compared with15% in animals of group 8 (high dose trastuzumab-MC-val-cit-PAB-MMAF).Elevation of liver enzymes AST and ALT, of bilirubin and the extent ofthrombocytopenia was most evident in animals treated withtrastuzumab-MC(Me)-val-cit-PAB-MMAF (groups 4 and 5) in a dose-dependentfashion; animals of group 5 (high dose group) showed on day 5 levels ofALT of approximately 10× the baseline value and platelets were reducedby approximately 90% at the time of necropsy.

Female Sprague Dawley Rats were also dosed at high levels (Example 19,High Dose study: Groups 2, 3, 4) with trastuzumab-MC-MMAF, and Vehiclecontrol (Group 1). Mild toxicity signals were observed, including adose-dependent elevation of liver enzymes ALT, AST and GGT. On day 5animals in the highest dose group showed a 2-fold elevation of ALT and a5-fold elevation of AST; GGT is also elevated (6U/L). Enzyme levels showa trend towards normalization on day 12. There was a mild granulocytosisin all three dose groups on day 5, the platelet count remainedessentially unchanged in all animals. Morphological changes were mild;animals treated at the 4210 μg/m² dose level (Group 2) showedunremarkable histology of liver, spleen, thymus, intestines and bonemarrow. Mildly increased apoptotic and mitotic activity was observed inthymus and liver, respectively in animals treated at the 5500 μg/m² doselevel (Group 3). The bone marrow was normocellular, but showed evidenceof granulocytic hyperplasia, which is consistent with the absolutegranulocytosis observed in the peripheral blood counts in these animals.Animals at the highest dose in group 4 showed qualitatively the samefeatures; the mitotic activity in the liver appears somewhat increasedcompared to animals in Group 3. Also, extramedullary hematopoiesis wasseen in spleen and liver.

EphB2R is a type 1 TM tyrosine kinase receptor with close homologybetween mouse and human, and is over-expressed in colorectal cancercells. 2H9 is an antibody against EphB2R. The naked antibody has noeffect on tumor growth, but 2H9-val-cit-MMAE killed EphB2R expressingcells and showed efficacy in a mouse xenograft model using CXF1103 humancolon tumors (Mao et al (2004) Cancer Res. 64:781-788). 2H9 and 7C2 areboth mouse IgG1 anti-HER2 antibodies. The toxicity profiles of2H9-MC-val-cit-PAB-MMAF (3.7 MMAF/Ab), 7C2-MC-val-cit-PAB-MMAF (4MMAF/Ab), and trastuzumab-MC-val-cit-PAB-MMAF (5.9 MMAF/Ab) werecompared. The differences in the structure of each immunoconjugate orthe drug portion of the immunoconjugate may affect the pharmacokineticsand ultimately the safety profile. The humanized trastuzumab antibodydoes not bind appreciably to rat tissue, and any toxicity would beconsidered non-specific.

Cynomolgus Monkey Toxicity/Safety

Similar to the rat toxicity/safety study, cynomolgus monkeys weretreated with ADC followed by liver enzyme measurements, and inspectionand analysis of the effects on various organs. Gross observationsincluded changes in body weights and signs of lesions and bleeding.Clinical pathology parameters (serum chemistry and hematology),histopathology, and necropsy were conducted on dosed animals (Example19).

The antibody drug conjugate, H-MC-vc-PAB-MMAE (H=trastuzumab linkedthrough cysteine) showed no evidence of liver toxicity at any of thedose levels tested. Peripheral blood granulocytes showed depletion aftera single dose of 1100 mg/m² with complete recovery 14 days post-dose.The antibody drug conjugate H-MC-vc-PAB-MMAF showed elevation of liverenzymes at 550 (transient) and 880 mg/m² dose level, no evidence ofgranulocytopenia, and a dose-dependent, transient (groups 2 & 3) declineof platelets.

Synthesis of the Compounds of the Invention

The Exemplary Compounds and Exemplary Conjugates can be made using thesynthetic procedures outlined below in FIGS. 25-36. As described in moredetail below, the Exemplary Compounds or Exemplary Conjugates can beconveniently prepared using a Linker having a reactive site for bindingto the Drug and Ligand. In one aspect, a Linker has a reactive sitewhich has an electrophilic group that is reactive to a nucleophilicgroup present on a Ligand, such as but not limited to an antibody.Useful nucleophilic groups on an antibody include but are not limitedto, sulfhydryl, hydroxyl and amino groups. The heteroatom of thenucleophilic group of an antibody is reactive to an electrophilic groupon a Linker and forms a covalent bond to a Linker unit. Usefulelectrophilic groups include, but are not limited to, maleimide andhaloacetamide groups. The electrophilic group provides a convenient sitefor antibody attachment.

In another embodiment, a Linker has a reactive site which has anucleophilic group that is reactive to an electrophilic group present onan antibody. Useful electrophilic groups on an antibody include, but arenot limited to, aldehyde and ketone carbonyl groups. The heteroatom of anucleophilic group of a Linker can react with an electrophilic group onan antibody and form a covalent bond to an antibody unit. Usefulnucleophilic groups on a Linker include, but are not limited to,hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazinecarboxylate, and arylhydrazide. The electrophilic group on an antibodyprovides a convenient site for attachment to a Linker.

Carboxylic acid functional groups and chloroformate functional groupsare also useful reactive sites for a Linker because they can react withsecondary amino groups of a Drug to form an amide linkage. Also usefulas a reactive site is a carbonate functional group on a Linker, such asbut not limited to p-nitrophenyl carbonate, which can react with anamino group of a Drug, such as but not limited to N-methyl valine, toform a carbamate linkage. Typically, peptide-based Drugs can be preparedby forming a peptide bond between two or more amino acids and/or peptidefragments. Such peptide bonds can be prepared, for example, according tothe liquid phase synthesis method (see E. Schröder and K. Lübke, “ThePeptides”, volume 1, pp 76-136, 1965, Academic Press) that is well knownin the field of peptide chemistry.

The synthesis of an illustrative Stretcher having an electrophilicmaleimide group is illustrated below in FIGS. 28 and 29. Generalsynthetic methods useful for the synthesis of a Linker are described inFIG. 30. FIG. 31 shows the construction of a Linker unit having aval-cit group, an electrophilic maleimide group and a PABself-immolative Spacer group. FIG. 32 depicts the synthesis of a Linkerhaving a phe-lys group, an electrophilic maleimide group, with andwithout the PAB self-immolative Spacer group. FIG. 33 presents a generaloutline for the synthesis of a Drug-Linker Compound, while FIG. 34presents an alternate route for preparing a Drug-Linker Compound. FIG.35 depicts the synthesis of a branched linker containing a BHMS group.FIG. 36 outlines the attachment of an antibody to a Drug-Linker Compoundto form a Drug-Linker-Antibody Conjugate, and FIG. 34 illustrates thesynthesis of Drug-Linker-Antibody Conjugates having, for example but notlimited to, 2 or 4 drugs per Antibody.

As described in more detail below, the Exemplary Conjugates areconveniently prepared using a Linker having two or more Reactive Sitesfor binding to the Drug and a Ligand. In one aspect, a Linker has aReactive site which has an electrophilic group that is reactive to anucleophilic group present on a Ligand, such as an antibody. Usefulnucleophilic groups on an antibody include but are not limited to,sulfhydryl, hydroxyl and amino groups. The heteroatom of thenucleophilic group of an antibody is reactive to an electrophilic groupon a Linker and forms a covalent bond to a Linker unit. Usefulelectrophilic groups include, but are not limited to, maleimide andhaloacetamide groups. The electrophilic group provides a convenient sitefor antibody attachment.

In another embodiment, a Linker has a Reactive site which has anucleophilic group that is reactive to an electrophilic group present ona Ligand, such as an antibody. Useful electrophilic groups on anantibody include, but are not limited to, aldehyde and ketone carbonylgroups. The heteroatom of a nucleophilic group of a Linker can reactwith an electrophilic group on an antibody and form a covalent bond toan antibody unit. Useful nucleophilic groups on a Linker include, butare not limited to, hydrazide, oxime, amino, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. Theelectrophilic group on an antibody provides a convenient site forattachment to a Linker.

Drug Moiety Synthesis

Typically, peptide-based Drugs can be prepared by forming a peptide bondbetween two or more amino acids and/or peptide fragments. Such peptidebonds can be prepared, for example, according to the liquid phasesynthesis method (see E. Schröder and K. Lüthke, “The Peptides”, volume1, pp 76-136, 1965, Academic Press) that is well known in the field ofpeptide chemistry.

The auristatin/dolastatin drug moieties may be prepared according to thegeneral methods of: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588;Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al.(1998) Anti-Cancer Drug Design 13:243-277; and Pettit et al. (1996) J.Chem. Soc. Perkin Trans. 15:859-863.

In one embodiment, a Drug is prepared by combining about astoichiometric equivalent of a dipeptide and a tripeptide, preferably ina one-pot reaction under suitable condensation conditions. This approachis illustrated in FIGS. 25-27, below.

FIG. 25 illustrates the synthesis of an N-terminal tripeptide unit Fwhich is a useful intermediate for the synthesis of the drug compoundsof Formula Ib.

As illustrated in FIG. 25, a protected amino acid A (where PG representsan amine protecting group, R⁴ is selected from hydrogen, C₁-C₈ alkyl,C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl, alkyl-aryl, alkyl-(C₃-C₈carbocycle), C₃-C₈ heterocycle, alkyl-(C₃-C₈ heterocycle) wherein R⁵ isselected from H and methyl; or R⁴ and R⁵ join, have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom hydrogen, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from2, 3, 4, 5 and 6, and form a ring with the carbon atom to which they areattached) is coupled to t-butyl ester B (where R⁶ is selected from —Hand —C₁-C₈ alkyl; and R⁷ is selected from hydrogen, C₁-C₈ alkyl, C₃-C₈carbocycle, —O—(C₁-C₈ alkyl), -aryl, alkyl-aryl, alkyl-(C₃-C₈carbocycle), C₃-C₈ heterocycle and alkyl-(C₃-C₈ heterocycle)) undersuitable coupling conditions, e.g., in the presence of PyBrop anddiisopropylethylamine, or using DCC (see, for example, Miyazaki, K. et.al. Chem. Pharm. Bull. 1995, 43(10), 1706-1718).

Suitable protecting groups PG, and suitable synthetic methods to protectan amino group with a protecting group are well known in the art. See,e.g., Greene, T. W. and Wuts, P. G. M., Protective Groups in OrganicSynthesis, 2nd Edition, 1991, John Wiley & Sons. Exemplary protectedamino acids A are PG-Ile and, particularly, PG-Val, while other suitableprotected amino acids include, without limitation: PG-cyclohexylglycine,PG-cyclohexylalanine, PG-aminocyclopropane-1-carboxylic acid,PG-aminoisobutyric acid, PG-phenylalanine, PG-phenylglycine, andPG-tert-butylglycine. Z is an exemplary protecting group. Fmoc isanother exemplary protecting group. An exemplary t-butyl ester B isdolaisoleuine t-butyl ester.

The dipeptide C can be purified, e.g., using chromatography, andsubsequently deprotected, e.g., using H₂ and 10% Pd—C in ethanol when PGis benzyloxycarbonyl, or using diethylamine for removal of an Fmocprotecting group. The resulting amine D readily forms a peptide bondwith an amino acid BB (wherein R¹ is selected from —H, —C₁-C₈ alkyl and—C₃-C₈ carbocycle; and R² is selected from —H and —C₁-C₈ alkyl; or R¹and R² join, have the formula —(CR^(a)R^(b))_(n)— wherein R^(a) andR^(b) are independently selected from —H, —C₁-C₈ alkyl and —C₃-C₈carbocycle and n is selected from 2, 3, 4, 5 and 6, and form a ring withthe nitrogen atom to which they are attached; and R³ is selected fromhydrogen, C₁-C₈ alkyl, C₃-C₈ carbocycle, —O—(C₁-C₈ alkyl), -aryl,alkyl-aryl, alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and alkyl-(C₃-C₈heterocycle)). N,N-Dialkyl amino acids are exemplary amino acids for BB,such as commercially available N,N-dimethyl valine. Other N,N-dialkylamino acids can be prepared by reductive bis-alkylation using knownprocedures (see, e.g., Bowman, R. E, Stroud, H. H J. Chem. Soc., 1950,1342-1340). Fmoc-Me-L-Val and Fmoc-Me-L-glycine are two exemplary aminoacids BB useful for the synthesis of N-monoalkyl derivatives. The amineD and the amino acid BB react to provide the tripeptide E using couplingreagent DEPC with triethylamine as the base. The C-terminus protectinggroup of E is subsequently deprotected using HCl to provide thetripeptide compound of formula F.

Illustrative DEPC coupling methodology and the PyBrop couplingmethodology shown in FIG. 25 are outlined below in General Procedure Aand General Procedure B, respectively. Illustrative methodology for thedeprotection of a Z-protected amine via catalytic hydrogenation isoutlined below in General Procedure C.

General Procedure A: Peptide Synthesis Using DEPC.

The N-protected or N,N-disubstituted amino acid or peptide D (1.0 eq.)and an amine BB (1.1 eq.) are diluted with an aprotic organic solvent,such as dichloromethane (0.1 to 0.5 M). An organic base such astriethylamine or diisopropylethylamine (1.5 eq.) is then added, followedby DEPC (1.1 eq.). The resulting solution is stirred, preferably underargon, for up to 12 hours while being monitored by HPLC or TLC. Thesolvent is removed in vacuo at room temperature, and the crude productis purified using, for example, HPLC or flash column chromatography(silica gel column). Relevant fractions are combined and concentrated invacuo to afford tripeptide E which is dried under vacuum overnight.

General Procedure B: Peptide Synthesis Using PyBrop.

The amino acid B (1.0 eq.), optionally having a carboxyl protectinggroup, is diluted with an aprotic organic solvent such asdichloromethane or DME to provide a solution of a concentration between0.5 and 1.0 mM, then diisopropylethylamine (1.5 eq.) is added. Fmoc-, orZ-protected amino acid A (1.1 eq.) is added as a solid in one portion,then PyBrop (1.2 eq.) is added to the resulting mixture. The reaction ismonitored by TLC or HPLC, followed by a workup procedure similar to thatdescribed in General Procedure A.

General Procedure C: Z-Removal Via Catalytic Hydrogenation.

Z-protected amino acid or peptide C is diluted with ethanol to provide asolution of a concentration between 0.5 and 1.0 mM in a suitable vessel,such as a thick-walled round bottom flask. 10% palladium on carbon isadded (5-10% w/w) and the reaction mixture is placed under a hydrogenatmosphere. Reaction progress is monitored using HPLC and is generallycomplete within 1-2 h. The reaction mixture is filtered through apre-washed pad of celite and the celite is again washed with a polarorganic solvent, such as methanol after filtration. The eluent solutionis concentrated in vacuo to afford a residue which is diluted with anorganic solvent, preferably toluene. The organic solvent is then removedin vacuo to afford the deprotected amine C.

FIG. 26 shows a method useful for making a C-terminal dipeptide offormula K and a method for coupling the dipeptide of formula K with thetripeptide of formula F to make drug compounds of Formula Ib.

The dipeptide K can be readily prepared by condensation of the modifiedamino acid Boc-Dolaproine G (see, for example, Pettit, G. R., et al.Synthesis, 1996, 719-725), with an amine of formula H using condensingagents well known for peptide chemistry, such as, for example, DEPC inthe presence of triethylamine, as shown in FIG. 25.

The dipeptide of formula K can then be coupled with a tripeptide offormula F using General Procedure D to make the Fmoc-protected drugcompounds of formula L which can be subsequently deprotected usingGeneral Procedure E in order to provide the drug compounds of formula(Ib).

General Procedure D: Drug Synthesis.

A mixture of dipeptide K (1.0 eq.) and tripeptide F (1 eq.) is dilutedwith an aprotic organic solvent, such as dichloromethane, to form a 0.1Msolution, then a strong acid, such as trifluoroacetic acid (1/2 v/v) isadded and the resulting mixture is stirred under a nitrogen atmospherefor two hours at 0° C. The reaction can be monitored using TLC or,preferably, HPLC. The solvent is removed in vacuo and the resultingresidue is azeotropically dried twice, preferably using toluene. Theresulting residue is dried under high vacuum for 12 h and then dilutedwith and aprotic organic solvent, such as dichloromethane. An organicbase such as triethylamine or diisopropylethylamine (1.5 eq.) is thenadded, followed by either PyBrop (1.2 eq.) or DEPC (1.2 eq.) dependingon the chemical functionality on the residue. The reaction mixture ismonitored by either TLC or HPLC and upon completion, the reaction issubjected to a workup procedure similar or identical to that describedin General Procedure A.

General Procedure E: Fmoc-Removal Using Diethylamine.

An Fmoc-protected Drug L is diluted with an aprotic organic solvent suchas dichloromethane and to the resulting solution is added diethylamine(1/2 v/v). Reaction progress is monitored by TLC or HPLC and istypically complete within 2 h. The reaction mixture is concentrated invacuo and the resulting residue is azeotropically dried, preferablyusing toluene, then dried under high vacuum to afford Drug Ib having adeprotected amino group.

FIG. 27 shows a method useful for making MMAF derivatives of Formula(Ib).

The dipeptide O can be readily prepared by condensation of the modifiedamino acid Boc-Dolaproine G (see, for example, Pettit, G. R., et al.Synthesis, 1996, 719-725), with a protected amino acid of formula Musing condensing agents well known for peptide chemistry, such as, forexample, DEPC in the presence of triethylamine, as shown in FIGS. 25 and26.

The dipeptide of formula O can then be coupled with a tripeptide offormula F using General Procedure D to make the Fmoc-protected MMAFcompounds of formula P which can be subsequently deprotected usingGeneral Procedure E in order to provide the MMAF drug compounds offormula (Ib).

Thus, the above methods are useful for making Drugs that can be used inthe present invention.

Drug Linker Synthesis

To prepare a Drug-Linker Compound of the present invention, the Drug isreacted with a reactive site on the Linker. In general, the Linker canhave the structure:

when both a Spacer unit (—Y—) and a Stretcher unit (-A-) are present.Alternately, the Linker can have the structure:

when the Spacer unit (—Y—) is absent.

The Linker can also have the structure:

when both the Stretcher unit (-A-) and the Spacer unit (—Y—) are absent.

The Linker can also have the structure:

when both the Amino Acid unit (W) and the Spacer Unit (Y) are absent.

In general, a suitable Linker has an Amino Acid unit linked to anoptional Stretcher Unit and an optional Spacer Unit. Reactive Site 1 ispresent at the terminus of the Spacer and Reactive site 2 is present atthe terminus of the Stretcher. If a Spacer unit is not present, thenReactive site 1 is present at the C-terminus of the Amino Acid unit.

In an exemplary embodiment of the invention, Reactive Site No. 1 isreactive to a nitrogen atom of the Drug, and Reactive Site No. 2 isreactive to a sulfhydryl group on the Ligand. Reactive Sites 1 and 2 canbe reactive to different functional groups.

In one aspect of the invention, Reactive Site No. 1 is

In another aspect of the invention, Reactive Site No. 1 is

In still another aspect of the invention, Reactive Site No. 1 is ap-nitrophenyl carbonate having the formula

In one aspect of the invention, Reactive Site No. 2 is a thiol-acceptinggroup. Suitable thiol-accepting groups include haloacetamide groupshaving the formula

wherein X represents a leaving group, preferably O-mesyl, O-tosyl, —Cl,—Br, or —I; or a maleimide group having the formula

Useful Linkers can be obtained via commercial sources, such as MolecularBiosciences Inc. (Boulder, Colo.), or prepared as summarized in FIGS.28-30.

In FIG. 28 X is —CH₂— or —CH₂OCH₂—; and n is an integer ranging eitherfrom 0-10 when X is —CH₂—; or 1-10 when X is —CH₂OCH₂—.

The method shown in FIG. 29 combines maleimide with a glycol underMitsunobu conditions to make a polyethylene glycol maleimide Stretcher(see for example, Walker, M. A. J. Org. Chem. 1995, 60, 5352-5),followed by installation of a p-nitrophenyl carbonate Reactive Sitegroup.

In FIG. 29 E is —CH₂— or —CH₂OCH₂—; and e is an integer ranging from0-8;

Alternatively, PEG-maleimide and PEG-haloacetamide stretchers can beprepared as described by Frisch, et al., Bioconjugate Chem. 1996, 7,180-186.

FIG. 30 illustrates a general synthesis of an illustrative Linker unitcontaining a maleimide Stretcher group and optionally a p-aminobenzylether self-immolative Spacer.

In FIG. 30 Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or-cyano; m is an integer ranging from 0-4; and n is an integer rangingfrom 0-10.

Useful Stretchers may be incorporated into a Linker using thecommercially available intermediates from Molecular Biosciences(Boulder, Colo.) described below by utilizing known techniques oforganic synthesis.

Stretchers of formula (IIIa) can be introduced into a Linker by reactingthe following intermediates with the N-terminus of an Amino Acid unit asdepicted in FIGS. 31 and 32:

where n is an integer ranging from 1-10 and T is —H or —SO₃Na;

where n is an integer ranging from 0-3;

Stretcher units of formula (IIIb) can be introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

-   -   where X is —Br or —I; and

Stretcher units of formula (IV) can be introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

Stretcher units of formula (Va) can be introduced into a Linker byreacting the following intermediates with the N-terminus of an AminoAcid unit:

Other useful Stretchers may be synthesized according to knownprocedures. Aminooxy Stretchers of the formula shown below can beprepared by treating alkyl halides with N-Boc-hydroxylamine according toprocedures described in Jones, D. S. et al., Tetrahedron Letters, 2000,41(10), 1531-1533; and Gilon, C. et al., Tetrahedron, 1967, 23(11),4441-4447.

wherein —R¹⁷— is selected from —C₁-C₁₀ alkylene-, —C₃-C₈ carbocyclo-,—O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀ alkylene-arylene-, -arylene-C₁-C₁₀alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈ carbocyclo)-, —(C₃-C₈carbocyclo)-C₁-C₁₀ alkylene-, —C₃-C₈ heterocyclo-, —C₁-C₁₀alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈ heterocyclo)-C₁-C₁₀ alkylene-,—(CH₂CH₂O)_(r)—, —(CH₂CH₂O)_(r)—CH₂—; and r is an integer ranging from1-10;Isothiocyanate Stretchers of the formula shown below may be preparedfrom isothiocyanatocarboxylic acid chlorides as described in Angew.Chem., 1975, 87(14):517.

wherein —R¹⁷— is as described herein.

FIG. 31 shows a method for obtaining of a val-cit dipeptide Linkerhaving a maleimide Stretcher and optionally a p-aminobenzylself-immolative Spacer.

In FIG. 31 Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4.

FIG. 32 illustrates the synthesis of a phe-lys(Mtr) dipeptide Linkerunit having a maleimide Stretcher unit and a p-aminobenzylself-immolative Spacer unit. Starting material AD (lys(Mtr)) iscommercially available (Bachem, Torrance, Calif.) or can be preparedaccording to Dubowchik, et al. Tetrahedron Letters (1997) 38:5257-60.

In FIG. 32 Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4.

As shown in FIG. 33, a Linker can be reacted with an amino group of aDrug Compound of Formula (Ib) to form a Drug-Linker Compound thatcontains an amide or carbamate group, linking the Drug unit to theLinker unit. When Reactive Site No. 1 is a carboxylic acid group, as inLinker AJ, the coupling reaction can be performed using HATU or PyBropand an appropriate amine base, resulting in a Drug-Linker Compound AK,containing an amide bond between the Drug unit and the Linker unit. WhenReactive Site No. 1 is a carbonate, as in Linker AL, the Linker can becoupled to the Drug using HOBt in a mixture of DMF/pyridine to provide aDrug-Linker Compound AM, containing a carbamate bond between the Drugunit and the Linker unit

Alternately, when Reactive Site No. 1 is a good leaving group, such asin Linker AN, the Linker can be coupled with an amine group of a Drugvia a nucleophilic substitution process to provide a Drug-LinkerCompound having an amine linkage (AO) between the Drug unit and theLinker unit.

Illustrative methods useful for linking a Drug to a Ligand to form aDrug-Linker Compound are depicted in FIG. 33 and are outlined in GeneralProcedures G-H.

General Procedure G: Amide Formation Using HATU.

A Drug (Ib) (1.0 eq.) and an N-protected Linker containing a carboxylicacid Reactive site (1.0 eq.) are diluted with a suitable organicsolvent, such as dichloromethane, and the resulting solution is treatedwith HATU (1.5 eq.) and an organic base, preferably pyridine (1.5 eq.).The reaction mixture is allowed to stir under an inert atmosphere,preferably argon, for 6 h, during which time the reaction mixture ismonitored using HPLC. The reaction mixture is concentrated and theresulting residue is purified using HPLC to yield the amide of formulaAK.

Procedure H: Carbamate Formation Using HOBt.

A mixture of a Linker AL having a p-nitrophenyl carbonate Reactive site(1.1 eq.) and Drug (Ib) (1.0 eq.) are diluted with an aprotic organicsolvent, such as DMF, to provide a solution having a concentration of50-100 mM, and the resulting solution is treated with HOBt (2.0 eq.) andplaced under an inert atmosphere, preferably argon. The reaction mixtureis allowed to stir for 15 min, then an organic base, such as pyridine(1/4 v/v), is added and the reaction progress is monitored using HPLC.The Linker is typically consumed within 16 h. The reaction mixture isthen concentrated in vacuo and the resulting residue is purified using,for example, HPLC to yield the carbamate AM.

An alternate method of preparing Drug-Linker Compounds is outlined inFIG. 34. Using the method of FIG. 34, the Drug is attached to a partialLinker unit (ZA, for example), which does not have a Stretcher unitattached. This provides intermediate AP, which has an Amino Acid unithaving an Fmoc-protected N-terminus. The Fmoc group is then removed andthe resulting amine intermediate AQ is then attached to a Stretcher unitvia a coupling reaction catalyzed using PyBrop or DEPC. The constructionof Drug-Linker Compounds containing either a bromoacetamide Stretcher ARor a PEG maleimide Stretcher AS is illustrated in FIG. 34.

In FIG. 34 Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4.

Methodology useful for the preparation of a Linker unit containing abranched spacer is shown in FIG. 35.

FIG. 35 illustrates the synthesis of a val-cit dipeptide linker having amaleimide Stretcher unit and a bis(4-hydroxymethyl)styrene (BHMS) unit.The synthesis of the BHMS intermediate (AW) has been improved fromprevious literature procedures (see International Publication No, WO9813059 to Firestone et al., and Crozet, M. P.; Archaimbault, G.;Vanelle, P.; Nouguier, R. Tetrahedron Lett. (1985) 26:5133-5134) andutilizes as starting materials, commercially available diethyl(4-nitrobenzyl)phosphonate (AT) and commercially available2,2-dimethyl-1,3-dioxan-5-one (AU). Linkers AY and BA can be preparedfrom intermediate AW using the methodology described in FIG. 29.

Dendritic Linkers

The linker may be a dendritic type linker for covalent attachment ofmore than one drug moiety through a branching, multifunctional linkermoiety to a Ligand, such as but not limited to an antibody (Sun et al.(2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al.(2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkerscan increase the molar ratio of drug to antibody, i.e. loading, which isrelated to the potency of the Drug-Linker-Ligand Conjugate. Thus, wherea cysteine engineered antibody bears only one reactive cytsteine thiolgroup, a multitude of drug moieties may be attached through a dendriticlinker.

The following exemplary embodiments of dendritic linker reagents allowup to nine nucleophilic drug moiety reagents to be conjugated byreaction with the chloroethyl nitrogen mustard functional groups:

Conjugation of Drug Moieties to Antibodies

FIG. 36 illustrates methodology useful for making Drug-Linker-Ligandconjugates having about 2 to about 4 drugs per antibody. An antibody istreated with a reducing agent, such as dithiothreitol (DTT) to reducesome or all of the cysteine disulfide residues to form highlynucleophilic cysteine thiol groups (—CH₂SH). The partially reducedantibody thus reacts with drug-linker compounds, or linker reagents,with electrophilic functional groups such as maleimide or α-halocarbonyl, according to the conjugation method at page 766 of Klussman,et al. (2004), Bioconjugate Chemistry 15(4):765-773.

For example, an antibody, e.g., AC10, dissolved in 500 mM sodium borateand 500 mM sodium chloride at pH 8.0 is treated with an excess of 100 mMdithiothreitol (DTT). After incubation at 37° C. for about 30 minutes,the buffer is exchanged by elution over Sephadex G25 resin and elutedwith PBS with 1 mM DTPA. The thiol/Ab value is checked by determiningthe reduced antibody concentration from the absorbance at 280 nm of thesolution and the thiol concentration by reaction with DTNB (Aldrich,Milwaukee, Wis.) and determination of the absorbance at 412 nm. Thereduced antibody dissolved in PBS is chilled on ice. The drug linker,e.g., MC-val-cit-PAB-MMAE in DMSO, dissolved in acetonitrile and waterat known concentration, is added to the chilled reduced antibody in PBS.After about one hour, an excess of maleimide is added to quench thereaction and cap any unreacted antibody thiol groups. The reactionmixture is concentrated by centrifugal ultrafiltration and the ADC,e.g., AC10-MC-vc-PAB-MMAE, is purified and desalted by elution throughG25 resin in PBS, filtered through 0.2 pin filters under sterileconditions, and frozen for storage.

A variety of antibody drug conjugates (ADC) were prepared, with avariety of linkers, and the drug moieties, MMAE and MMAF. The followingtable is an exemplary group of ADC which were prepared following theprotocol of Example 27, and characterized by HPLC and drug loadingassay.

Target isolated amount drug/Ab (antigen) ADC (mg) ratio 0772P16E12-MC-vc-PAB-MMAE 1.75 4 0772P 11D10-MC-vc-PAB-MMAE 46.8 4.4 0772P11D10-MC-vc-PAB-MMAF 54.5 3.8 Brevican Brevican-MC-MMAF 2 6 BrevicanBrevican-MC-vc-MMAF 2 6 Brevican Brevican-MC-vc-PAB-MMAF 1.4 6 CD21CD21-MC-vc-PAB-MMAE 38.1 4.3 CD21 CD21-MC-vc-PAB-MMAF 43 4.1 CRIPTO11F4-MC-vc-PAB-MMAF 6 4.8 CRIPTO 25G8-MC-vc-PAB-MMAF 7.4 4.7 E1612G12-MC-vc-PAB-MMAE 2.3 4.6 E16 3B5-MC-vc-PAB-MMAE 2.9 4.6 E1612B9-MC-vc-PAB-MMAE 1.4 3.8 E16 12B9-MC-vc-PAB-MMAE 5.1 4 E1612G12-MC-vc-PAB-MMAE 3 4.6 E16 3B5-MC-vc-PAB-MMAE 4.8 4.1 E163B5-MC-vc-PAB-MMAF 24.7 4.4 EphB2R 2H9-MC-vc-PAB-MMAE 29.9 7.1 EphB2R2H9-MC-fk-PAB-MMAE 25 7.5 EphB2R 2H9-MC-vc-PAB-MMAE 175 4.1 EphB2R2H9-MC-vc-PAB-MMAF 150 3.8 EphB2R 2H9-MC-vc-PAB-MMAF 120 3.7 EphB2R2H9-MC-vc-PAB-MMAE 10.7 4.4 IL-20Ra IL20Ra-fk-MMAE 26 6.7 IL-20RaIL20Ra-vc-MMAE 27 7.3 EphB2 IL8-MC-vc-PAB-MMAE 251 3.7 MDP MDP-vc-MMAE32 MPF 19C3-vc-MMAE 1.44 6.5 MPF 7D9-vc-MMAE 4.3 3.8 MPF 19C3-vc-MMAE7.9 3 MPF 7D9-MC-vc-PAB-MMAF 5 4.3 Napi3b 10H1-vc-MMAE 4.5 4.6 Napi3b4C9-vc-MMAE 3.0 5.4 Napi3b 10H1-vc-MMAE 4.5 4.8 Napi3b 10H1-vc-MMAF 6.54 NCA 3E6-MC-fk-PAB-MMAE 49.6 5.4 NCA 3E6-MC-vc-PAB-MMAE 56.2 6.4 PSCAPSCA-fk-MMAE 51.7 8.9 PSCA PSCA-vc-MMAE 61.1 8.6 Napi3b10H1-MC-vc-PAB-MMAE 75 4.2 Napi3b 10H1-MC-vc-PAB-MMAF 95 4.4 Napi3b10H1-MC-MMAF 92 4 EphB2R 2H9-MC-vc-PAB-MMAE 79 5 EphB2R 2H9-MC-MMAF 924.9 0772P 11D10(Fc chimera)-MC-vc-PAB- 79 4.3 MMAE 0772P 11D10(Fcchimera)-MC-vc-PAB- 70 4.5 MMAF 0772P 11D10(Fc chimera)-MC-MMAF 23 4.5Brevican 6D2-MC-vc-PAB-MMAF 0.3 4.5 Brevican 6D2-MC-MMAF 0.36 4.5 EphB2R2H9(Fc chimera)-MC-vc-PAB- 1983 4.3 MMAE E16 12B9-MC-vc-PAB-MMAE 14.14.6 E16 12B9-MC-vc-PAB-MMAF 16.4 4.5 E16 12G12-MC-vc-PAB-MMAE 10.5 4.1E16 12G12-MC-vc-PAB-MMAF 10.2 3.8 E16 3B5-MC-vc-PAB-MMAE 58.6 3.8 E163B5-MC-vc-PAB-MMAF 8 3.1 0772P 11D10(Fc chimera)-MC-vc-PAB- 340 3.9 MMAESteap1 (Steap1-92)-MC-vc-PAB-MMAE 3.5 4 Steap1(Steap1-92)-MC-vc-PAB-MMAF 4.7 4 Steap1 (Steap1-120)-MC-vc-PAB- 2 4 MMAESteap1 (Steap1-120)-MC-vc-PAB-MMAF 2.3 4 E16 3B5-MC-vc-PAB-MMAF 52.2 4.5

Compositions and Methods of Administration

In other embodiments, described is a composition including an effectiveamount of an Exemplary Compound and/or Exemplary Conjugate and apharmaceutically acceptable carrier or vehicle. For convenience, theDrug units and Drug-Linker Compounds can be referred to as ExemplaryCompounds, while Drug-Ligand Conjugates and Drug-Linker-LigandConjugates can be referred to as Exemplary Conjugates. The compositionsare suitable for veterinary or human administration.

The present compositions can be in any form that allows for thecomposition to be administered to a patient. For example, thecomposition can be in the form of a solid, liquid or gas (aerosol).Typical routes of administration include, without limitation, oral,topical, parenteral, sublingual, rectal, vaginal, ocular, intra-tumor,and intranasal. Parenteral administration includes subcutaneousinjections, intravenous, intramuscular, intrasternal injection orinfusion techniques. In one aspect, the compositions are administeredparenterally. In yet another aspect, the Exemplary Compounds and/or theExemplary Conjugates or compositions are administered intravenously.

Pharmaceutical compositions can be formulated so as to allow anExemplary Compound and/or Exemplary Conjugate to be bioavailable uponadministration of the composition to a patient. Compositions can takethe form of one or more dosage units, where for example, a tablet can bea single dosage unit, and a container of an Exemplary Compound and/orExemplary Conjugate in aerosol form can hold a plurality of dosageunits.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the Exemplary Compound or ExemplaryConjugate, the manner of administration, and the composition employed.

The pharmaceutically acceptable carrier or vehicle can be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid. In addition, the carrier(s) can begaseous or particulate, so as to provide an aerosol composition usefulin, e.g., inhalatory administration.

When intended for oral administration, the composition is preferably insolid or liquid form, where semi-solid, semi-liquid, suspension and gelforms are included within the forms considered herein as either solid orliquid.

As a solid composition for oral administration, the composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such asstarch, lactose or dextrins, disintegrating agents such as alginic acid,sodium alginate, Primogel, corn starch and the like; lubricants such asmagnesium stearate or Sterotex; glidants such as colloidal silicondioxide; sweetening agents such as sucrose or saccharin, a flavoringagent such as peppermint, methyl salicylate or orange flavoring, and acoloring agent.

When the composition is in the form of a capsule, e.g., a gelatincapsule, it can contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition can be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid can be useful for oraladministration or for delivery by injection. When intended for oraladministration, a composition can comprise one or more of a sweeteningagent, preservatives, dye/colorant and flavor enhancer. In a compositionfor administration by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent can also be included.

The liquid compositions, whether they are solutions, suspensions orother like form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or digylcerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. A parenteral composition can be enclosed inampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is an exemplaryadjuvant. An injectable composition is preferably sterile.

The amount of the Exemplary Compound and/or Exemplary Conjugate that iseffective in the treatment of a particular disorder or condition willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro or in vivo assayscan optionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the compositions will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances.

The compositions comprise an effective amount of an Exemplary Compoundand/or Exemplary Conjugate such that a suitable dosage will be obtained.Typically, this amount is at least about 0.01% of an Exemplary Compoundand/or Exemplary Conjugate by weight of the composition. When intendedfor oral administration, this amount can be varied to range from about0.1% to about 80% by weight of the composition. In one aspect, oralcompositions can comprise from about 4% to about 50% of the ExemplaryCompound and/or Exemplary Conjugate by weight of the composition. In yetanother aspect, present compositions are prepared so that a parenteraldosage unit contains from about 0.01% to about 2% by weight of theExemplary Compound and/or Exemplary Conjugate.

For intravenous administration, the composition can comprise from about0.01 to about 100 mg of an Exemplary Compound and/or Exemplary Conjugateper kg of the animal's body weight. In one aspect, the composition caninclude from about 1 to about 100 mg of an Exemplary Compound and/orExemplary Conjugate per kg of the animal's body weight. In anotheraspect, the amount administered will be in the range from about 0.1 toabout 25 mg/kg of body weight of the Exemplary Compound and/or ExemplaryConjugate.

Generally, the dosage of an Exemplary Compound and/or ExemplaryConjugate administered to a patient is typically about 0.01 mg/kg toabout 2000 mg/kg of the animal's body weight. In one aspect, the dosageadministered to a patient is between about 0.01 mg/kg to about 10 mg/kgof the animal's body weight, in another aspect, the dosage administeredto a patient is between about 0.1 mg/kg and about 250 mg/kg of theanimal's body weight, in yet another aspect, the dosage administered toa patient is between about 0.1 mg/kg and about 20 mg/kg of the animal'sbody weight, in yet another aspect the dosage administered is betweenabout 0.1 mg/kg to about 10 mg/kg of the animal's body weight, and inyet another aspect, the dosage administered is between about 1 mg/kg toabout 10 mg/kg of the animal's body weight.

The Exemplary Compounds and/or Exemplary Conjugate or compositions canbe administered by any convenient route, for example by infusion orbolus injection, by absorption through epithelial or mucocutaneouslinings (e.g., oral mucosa, rectal and intestinal mucosa, etc.).Administration can be systemic or local. Various delivery systems areknown, e.g., encapsulation in liposomes, microparticles, microcapsules,capsules, etc., and can be used to administer an Exemplary Compoundand/or Exemplary Conjugate or composition. In certain embodiments, morethan one Exemplary Compound and/or Exemplary Conjugate or composition isadministered to a patient.

In specific embodiments, it can be desirable to administer one or moreExemplary Compounds and/or Exemplary Conjugate or compositions locallyto the area in need of treatment. This can be achieved, for example, andnot by way of limitation, by local infusion during surgery; topicalapplication, e.g., in conjunction with a wound dressing after surgery;by injection; by means of a catheter; by means of a suppository; or bymeans of an implant, the implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. In one embodiment, administration can be by direct injectionat the site (or former site) of a cancer, tumor or neoplastic orpre-neoplastic tissue. In another embodiment, administration can be bydirect injection at the site (or former site) of a manifestation of anautoimmune disease.

In certain embodiments, it can be desirable to introduce one or moreExemplary Compounds and/or Exemplary Conjugate or compositions into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection. Intraventricular injection can be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir.

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent, or viaperfusion in a fluorocarbon or synthetic pulmonary surfactant.

In yet another embodiment, the Exemplary Compounds and/or ExemplaryConjugate or compositions can be delivered in a controlled releasesystem, such as but not limited to, a pump or various polymericmaterials can be used. In yet another embodiment, a controlled-releasesystem can be placed in proximity of the target of the ExemplaryCompounds and/or Exemplary Conjugate or compositions, e.g., the brain,thus requiring only a fraction of the systemic dose (see, e.g., Goodson,in Medical Applications of Controlled Release, supra, vol. 2, pp.115-138 (1984)). Other controlled-release systems discussed in thereview by Langer (Science 249:1527-1533 (1990)) can be used.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich an Exemplary Compound and/or Exemplary Conjugate is administered.Such pharmaceutical carriers can be liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.The carriers can be saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea, and the like. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents can be used. Inone embodiment, when administered to a patient, the Exemplary Compoundand/or Exemplary Conjugate or compositions and pharmaceuticallyacceptable carriers are sterile. Water is an exemplary carrier when theExemplary Compounds and/or Exemplary Conjugates are administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The present compositions, if desired, canalso contain minor amounts of wetting or emulsifying agents, or pHbuffering agents.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, aerosols, sprays, suspensions, or any other form suitable foruse. Other examples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

In an embodiment, the Exemplary Compounds and/or Exemplary Conjugatesare formulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to animals,particularly human beings. Typically, the carriers or vehicles forintravenous administration are sterile isotonic aqueous buffersolutions. Where necessary, the compositions can also include asolubilizing agent. Compositions for intravenous administration canoptionally comprise a local anesthetic such as lignocaine to ease painat the site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent. Where an Exemplary Compound and/or Exemplary Conjugateis to be administered by infusion, it can be dispensed, for example,with an infusion bottle containing sterile pharmaceutical grade water orsaline. Where the Exemplary Compound and/or Exemplary Conjugate isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients can be mixed prior toadministration.

Compositions for oral delivery can be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups, or elixirs, for example. Orally administered compositions cancontain one or more optionally agents, for example, sweetening agentssuch as fructose, aspartame or saccharin; flavoring agents such aspeppermint, oil of wintergreen, or cherry; coloring agents; andpreserving agents, to provide a pharmaceutically palatable preparation.Moreover, where in tablet or pill form, the compositions can be coatedto delay disintegration and absorption in the gastrointestinal tractthereby providing a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving compound are also suitable for orally administered compounds. Inthese later platforms, fluid from the environment surrounding thecapsule is imbibed by the driving compound, which swells to displace theagent or agent composition through an aperture. These delivery platformscan provide an essentially zero order delivery profile as opposed to thespiked profiles of immediate release formulations. A time-delay materialsuch as glycerol monostearate or glycerol stearate can also be used.

The compositions can be intended for topical administration, in whichcase the carrier may be in the form of a solution, emulsion, ointment orgel base. If intended for transdermal administration, the compositioncan be in the form of a transdermal patch or an iontophoresis device.Topical formulations can comprise a concentration of an ExemplaryCompound and/or Exemplary Conjugate of from about 0.05% to about 50% w/v(weight per unit volume of composition), in another aspect, from 0.1% to10% w/v.

The composition can be intended for rectal administration, in the form,e.g., of a suppository which will melt in the rectum and release theExemplary Compound and/or Exemplary Conjugate.

The composition can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

The compositions can consist of gaseous dosage units, e.g., it can be inthe form of an aerosol. The term aerosol is used to denote a variety ofsystems ranging from those of colloidal nature to systems consisting ofpressurized packages. Delivery can be by a liquefied or compressed gasor by a suitable pump system that dispenses the active ingredients.

Whether in solid, liquid or gaseous form, the present compositions caninclude a pharmacological agent used in the treatment of cancer, anautoimmune disease or an infectious disease.

Therapeutic Uses of the Exemplary Conjugates

The Exemplary Compounds and/or Exemplary Conjugates are useful fortreating cancer, an autoimmune disease or an infectious disease in apatient.

Treatment of Cancer

The Exemplary Compounds and/or Exemplary Conjugates are useful forinhibiting the multiplication of a tumor cell or cancer cell, causingapoptosis in a tumor or cancer cell, or for treating cancer in apatient. The Exemplary Compounds and/or Exemplary Conjugates can be usedaccordingly in a variety of settings for the treatment of animalcancers. The Drug-Linker-Ligand Conjugates can be used to deliver a Drugor Drug unit to a tumor cell or cancer cell. Without being bound bytheory, in one embodiment, the Ligand unit of an Exemplary Conjugatebinds to or associates with a cancer-cell or a tumor-cell-associatedantigen, and the Exemplary Conjugate can be taken up inside a tumor cellor cancer cell through receptor-mediated endocytosis. The antigen can beattached to a tumor cell or cancer cell or can be an extracellularmatrix protein associated with the tumor cell or cancer cell. Onceinside the cell, one or more specific peptide sequences within theLinker unit are hydrolytically cleaved by one or more tumor-cell orcancer-cell-associated proteases, resulting in release of a Drug or aDrug-Linker Compound. The released Drug or Drug-Linker Compound is thenfree to migrate within the cell and induce cytotoxic or cytostaticactivities. In an alternative embodiment, the Drug or Drug unit iscleaved from the Exemplary Conjugate outside the tumor cell or cancercell, and the Drug or Drug-Linker Compound subsequently penetrates thecell.

In one embodiment, the Ligand unit binds to the tumor cell or cancercell.

In another embodiment, the Ligand unit binds to a tumor cell or cancercell antigen which is on the surface of the tumor cell or cancer cell.

In another embodiment, the Ligand unit binds to a tumor cell or cancercell antigen which is an extracellular matrix protein associated withthe tumor cell or cancer cell.

The specificity of the Ligand unit for a particular tumor cell or cancercell can be important for determining those tumors or cancers that aremost effectively treated. For example, Exemplary Conjugates having aBR96 Ligand unit can be useful for treating antigen positive carcinomasincluding those of the lung, breast, colon, ovaries, and pancreas.Exemplary Conjugates having an Anti-CD30 or an anti-CD40 Ligand unit canbe useful for treating hematologic malignancies.

Other particular types of cancers that can be treated with ExemplaryConjugates include, but are not limited to, those disclosed in Table 3.

TABLE 3 Solid tumors, including but not limited to: fibrosarcomamyxosarcoma liposarcoma chondrosarcoma osteogenic sarcoma chordomaangiosarcoma endotheliosarcoma lymphangiosarcomalymphangioendotheliosarcoma synovioma mesothelioma Ewing's tumorleiomyosarcoma rhabdomyosarcoma colon cancer colorectal cancer kidneycancer pancreatic cancer bone cancer breast cancer ovarian cancerprostate cancer esophogeal cancer stomach cancer oral cancer nasalcancer throat cancer squamous cell carcinoma basal cell carcinomaadenocarcinoma sweat gland carcinoma sebaceous gland carcinoma papillarycarcinoma papillary adenocarcinomas cystadenocarcinoma medullarycarcinoma bronchogenic carcinoma renal cell carcinoma hepatoma bile ductcarcinoma choriocarcinoma seminoma embryonal carcinoma Wilms′ tumorcervical cancer uterine cancer testicular cancer small cell lungcarcinoma bladder carcinoma lung cancer epithelial carcinoma gliomaglioblastoma multiforme astrocytoma medulloblastoma craniopharyngiomaependymoma pinealoma hemangioblastoma acoustic neuroma oligodendrogliomameningioma skin cancer melanoma neuroblastoma retinoblastoma blood-bornecancers, including but not limited to: acute lymphoblastic leukemia“ALL” acute lymphoblastic B-cell leukemia acute lymphoblastic T-cellleukemia acute myeloblastic leukemia “AML” acute promyelocytic leukemia“APL” acute monoblastic leukemia acute erythroleukemic leukemia acutemegakaryoblastic leukemia acute myelomonocytic leukemia acutenonlymphocyctic leukemia acute undifferentiated leukemia chronicmyelocytic leukemia “CML” chronic lymphocytic leukemia “CLL” hairy cellleukemia multiple myeloma acute and chronic leukemias: lymphoblasticmyelogenous lymphocytic myelocytic leukemias Lymphomas: Hodgkin'sdisease non-Hodgkin's Lymphoma Multiple myeloma Waldenström'smacroglobulinemia Heavy chain disease Polycythemia vera

The Exemplary Conjugates provide conjugation-specific tumor or cancertargeting, thus reducing general toxicity of these compounds. The Linkerunits stabilize the Exemplary Conjugates in blood, yet are cleavable bytumor-specific proteases within the cell, liberating a Drug.

Multi-Modality Therapy for Cancer

Cancers, including, but not limited to, a tumor, metastasis, or otherdisease or disorder characterized by uncontrolled cell growth, can betreated or prevented by administration of an Exemplary Conjugate and/oran Exemplary Compound.

In other embodiments, methods for treating or preventing cancer areprovided, including administering to a patient in need thereof aneffective amount of an Exemplary Conjugate and a chemotherapeutic agent.In one embodiment the chemotherapeutic agent is that with whichtreatment of the cancer has not been found to be refractory. In anotherembodiment, the chemotherapeutic agent is that with which the treatmentof cancer has been found to be refractory. The Exemplary Conjugates canbe administered to a patient that has also undergone surgery astreatment for the cancer.

In one embodiment, the additional method of treatment is radiationtherapy.

In a specific embodiment, the Exemplary Conjugate is administeredconcurrently with the chemotherapeutic agent or with radiation therapy.In another specific embodiment, the chemotherapeutic agent or radiationtherapy is administered prior or subsequent to administration of anExemplary Conjugates, in one aspect at least an hour, five hours, 12hours, a day, a week, a month, in further aspects several months (e.g.,up to three months), prior or subsequent to administration of anExemplary Conjugate.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the chemotherapeutic agents listed in Table4 can be administered. With respect to radiation, any radiation therapyprotocol can be used depending upon the type of cancer to be treated.For example, but not by way of limitation, x-ray radiation can beadministered; in particular, high-energy megavoltage (radiation ofgreater that 1 MeV energy) can be used for deep tumors, and electronbeam and orthovoltage x-ray radiation can be used for skin cancers.Gamma-ray emitting radioisotopes, such as radioactive isotopes ofradium, cobalt and other elements, can also be administered.

Additionally, methods of treatment of cancer with an Exemplary Compoundand/or Exemplary Conjugate are provided as an alternative tochemotherapy or radiation therapy where the chemotherapy or theradiation therapy has proven or can prove too toxic, e.g., results inunacceptable or unbearable side effects, for the subject being treated.The animal being treated can, optionally, be treated with another cancertreatment such as surgery, radiation therapy or chemotherapy, dependingon which treatment is found to be acceptable or bearable.

The Exemplary Compounds and/or Exemplary Conjugates can also be used inan in vitro or ex vivo fashion, such as for the treatment of certaincancers, including, but not limited to leukemias and lymphomas, suchtreatment involving autologous stem cell transplants. This can involve amulti-step process in which the animal's autologous hematopoietic stemcells are harvested and purged of all cancer cells, the animal'sremaining bone-marrow cell population is then eradicated via theadministration of a high dose of an Exemplary Compound and/or ExemplaryConjugate with or without accompanying high dose radiation therapy, andthe stem cell graft is infused back into the animal. Supportive care isthen provided while bone marrow function is restored and the animalrecovers.

Multi-Drug Therapy for Cancer

Methods for treating cancer including administering to a patient in needthereof an effective amount of an Exemplary Conjugate and anothertherapeutic agent that is an anti-cancer agent are disclosed. Suitableanticancer agents include, but are not limited to, methotrexate, taxol,L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine,cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin,mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards,cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins,bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin,plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine,vinorelbine, paclitaxel, and docetaxel. In one aspect, the anti-canceragent includes, but is not limited to, a drug listed in Table 4.

TABLE 4 Alkylating agents Nitrogen mustards: cyclophosphamide ifosfamidetrofosfamide chlorambucil melphalan Nitrosoureas: carmustine (BCNU)lomustine (CCNU) Alkylsulphonates busulfan treosulfan Triazenes:decarbazine Platinum containing compounds: cisplatin carboplatin PlantAlkaloids Vinca alkaloids: vincristine vinblastine vindesine vinorelbineTaxoids: paclitaxel docetaxol DNA Topoisomerase InhibitorsEpipodophyllins: etoposide teniposide topotecan 9-aminocamptothecincamptothecin crisnatol mitomycins: mitomycin C Anti-metabolitesAnti-folates: DHFR inhibitors: methotrexate trimetrexate IMPdehydrogenase Inhibitors: mycophenolic acid tiazofurin ribavirin EICARRibonucleotide reductase Inhibitors: hydroxyurea deferoxamine Pyrimidineanalogs: Uracil analogs 5-Fluorouracil floxuridine doxifluridineratitrexed Cytosine analogs cytarabine (ara C) cytosine arabinosidefludarabine Purine analogs: mercaptopurine thioguanine Hormonaltherapies: Receptor antagonists: Anti-estrogen tamoxifen raloxifenemegestrol LHRH agonists: goscrclin leuprolide acetate Anti-androgens:flutamide bicalutamide Retinoids/Deltoids Vitamin D3 analogs: EB 1089 CB1093 KH 1060 Photodynamic therapies: vertoporfin (BPD-MA) phthalocyaninephotosensitizer Pc4 demethoxy-hypocrellin A (2BA-2-DMHA) Cytokines:Interferon-α Interferon-γ tumor necrosis factor Others: GemcitabineVelcade Revamid Thalamid Isoprenylation inhibitors: LovastatinDopaminergic neurotoxins: 1-methyl-4-phenylpyridinium ion Cell cycleinhibitors: staurosporine Actinomycins: Actinomycin D dactinomycinBleomycins: bleomycin A2 bleomycin B2 peplomycin Anthracyclines:daunorubicin Doxorubicin (adriamycin) idarubicin epirubicin pirarubicinzorubicin mtoxantrone MDR inhibitors: verapamil Ca²⁺ATPase inhibitors:thapsigargin

Treatment of Autoimmune Diseases

The Exemplary Conjugates are useful for killing or inhibiting thereplication of a cell that produces an autoimmune disease or fortreating an autoimmune disease. The Exemplary Conjugates can be usedaccordingly in a variety of settings for the treatment of an autoimmunedisease in a patient. The Drug-Linker-Ligand Conjugates can be used todeliver a Drug to a target cell. Without being bound by theory, in oneembodiment, the Drug-Linker-Ligand Conjugate associates with an antigenon the surface of a target cell, and the Exemplary Conjugate is thentaken up inside a target-cell through receptor-mediated endocytosis.Once inside the cell, one or more specific peptide sequences within theLinker unit are enzymatically or hydrolytically cleaved, resulting inrelease of a Drug. The released Drug is then free to migrate in thecytosol and induce cytotoxic or cytostatic activities. In an alternativeembodiment, the Drug is cleaved from the Exemplary Conjugate outside thetarget cell, and the Drug subsequently penetrates the cell.

In one embodiment, the Ligand unit binds to an autoimmune antigen. Inone aspect, the antigen is on the surface of a cell involved in anautoimmune condition.

In another embodiment, the Ligand unit binds to an autoimmune antigenwhich is on the surface of a cell.

In one embodiment, the Ligand binds to activated lymphocytes that areassociated with the autoimmune disease state.

In a further embodiment, the Exemplary Conjugates kill or inhibit themultiplication of cells that produce an autoimmune antibody associatedwith a particular autoimmune disease.

Particular types of autoimmune diseases that can be treated with theExemplary Conjugates include, but are not limited to, Th2 lymphocyterelated disorders (e.g., atopic dermatitis, atopic asthma,rhinoconjunctivitis, allergic rhinitis, Omenn's syndrome, systemicsclerosis, and graft versus host disease); Th1 lymphocyte-relateddisorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis,Sjorgren's syndrome, Hashimoto's thyroiditis, Grave's disease, primarybiliary cirrhosis, Wegener's granulomatosis, and tuberculosis);activated B lymphocyte-related disorders (e.g., systemic lupuserythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type Idiabetes); and those disclosed in Table 5.

TABLE 5 Active Chronic Hepatitis Addison's Disease Allergic AlveolitisAllergic Reaction Allergic Rhinitis Alport's Syndrome AnaphlaxisAnkylosing Spondylitis Anti-phosholipid Syndrome Arthritis AscariasisAspergillosis Atopic Allergy Atropic Dermatitis Atropic RhinitisBehcet's Disease Bird-Fancier's Lung Bronchial Asthma Caplan's SyndromeCardiomyopathy Celiac Disease Chagas' Disease Chronic GlomerulonephritisCogan's Syndrome Cold Agglutinin Disease Congenital Rubella InfectionCREST Syndrome Crohn's Disease Cryoglobulinemia Cushing's SyndromeDermatomyositis Discoid Lupus Dressler's Syndrome Eaton-Lambert SyndromeEchovirus Infection Encephalomyelitis Endocrine opthalmopathyEpstein-Barr Virus Infection Equine Heaves Erythematosis Evan's SyndromeFelty's Syndrome Fibromyalgia Fuch's Cyclitis Gastric AtrophyGastrointestinal Allergy Giant Cell Arteritis GlomerulonephritisGoodpasture's Syndrome Graft v. Host Disease Graves' DiseaseGuillain-Barre Disease Hashimoto's Thyroiditis Hemolytic AnemiaHenoch-Schonlein Purpura Idiopathic Adrenal Atrophy Idiopathic PulmonaryFibritis IgA Nephropathy Inflammatory Bowel Diseases Insulin-dependentDiabetes Mellitus Juvenile Arthritis Juvenile Diabetes Mellitus (Type I)Lambert-Eaton Syndrome Laminitis Lichen Planus Lupoid Hepatitis LupusLymphopenia Meniere's Disease Mixed Connective Tissue Disease MultipleSclerosis Myasthenia Gravis Pernicious Anemia Polyglandular SyndromesPresenile Dementia Primary Agammaglobulinemia Primary Biliary CirrhosisPsoriasis Psoriatic Arthritis Raynauds Phenomenon Recurrent AbortionReiter's Syndrome Rheumatic Fever Rheumatoid Arthritis Sampter'sSyndrome Schistosomiasis Schmidt's Syndrome Scleroderma Shulman'sSyndrome Sjorgen's Syndrome Stiff-Man Syndrome Sympathetic OphthalmiaSystemic Lupus Erythematosis Takayasu's Arteritis Temporal ArteritisThyroiditis Thrombocytopenia Thyrotoxicosis Toxic Epidermal NecrolysisType B Insulin Resistance Type I Diabetes Mellitus Ulcerative ColitisUveitis Vitiligo Waldenstrom's Macroglobulemia Wegener's Granulomatosis

Multi-Drug Therapy of Autoimmune Diseases

Methods for treating an autoimmune disease are also disclosed includingadministering to a patient in need thereof an effective amount of anExemplary Conjugate and another therapeutic agent known for thetreatment of an autoimmune disease. In one embodiment, theanti-autoimmune disease agent includes, but is not limited to, agentslisted in Table 6.

TABLE 6 cyclosporine cyclosporine A mycophenylate mofetil sirolimustacrolimus enanercept prednisone azathioprine methotrexatecyclophosphamide prednisone aminocaproic acid chloroquinehydroxychloroquine hydrocortisone dexamethasone chlorambucil DHEAdanazol bromocriptine meloxicam infliximab

Treatment of Infectious Diseases

The Exemplary Conjugates are useful for killing or inhibiting themultiplication of a cell that produces an infectious disease or fortreating an infectious disease. The Exemplary Conjugates can be usedaccordingly in a variety of settings for the treatment of an infectiousdisease in a patient. The Drug-Linker-Ligand Conjugates can be used todeliver a Drug to a target cell. In one embodiment, the Ligand unitbinds to the infectious disease cell.

In one embodiment, the Conjugates kill or inhibit the multiplication ofcells that produce a particular infectious disease.

Particular types of infectious diseases that can be treated with theExemplary Conjugates include, but are not limited to, those disclosed inTable 7.

TABLE 7 Bacterial Diseases: Diphtheria Pertussis Occult BacteremiaUrinary Tract Infection Gastroenteritis Cellulitis EpiglottitisTracheitis Adenoid Hypertrophy Retropharyngeal Abcess Impetigo EcthymaPneumonia Endocarditis Septic Arthritis Pneumococcal PeritonitisBactermia Meningitis Acute Purulent Meningitis Urethritis CervicitisProctitis Pharyngitis Salpingitis Epididymitis Gonorrhea SyphilisListeriosis Anthrax Nocardiosis Salmonella Typhoid Fever DysenteryConjunctivitis Sinusitis Brucellosis Tullaremia Cholera Bubonic PlagueTetanus Necrotizing Enteritis Actinomycosis Mixed Anaerobic InfectionsSyphilis Relapsing Fever Leptospirosis Lyme Disease Rat Bite FeverTuberculosis Lymphadenitis Leprosy Chlamydia Chlamydial PneumoniaTrachoma Inclusion Conjunctivitis Systemic Fungal Diseases:Histoplamosis Coccidiodomycosis Blastomycosis SporotrichosisCryptococcsis Systemic Candidiasis Aspergillosis Mucormycosis MycetomaChromomycosis Rickettsial Diseases: Typhus Rocky Mountain Spotted FeverEhrlichiosis Eastern Tick-Borne Rickettsioses Rickettsialpox Q FeverBartonellosis Parasitic Diseases: Malaria Babesiosis African SleepingSickness Chagas' Disease Leishmaniasis Dum-Dum Fever ToxoplasmosisMeningoencephalitis Keratitis Entamebiasis Giardiasis CryptosporidiasisIsosporiasis Cyclosporiasis Microsporidiosis Ascariasis WhipwormInfection Hookworm Infection Threadworm Infection Ocular Larva MigransTrichinosis Guinea Worm Disease Lymphatic Filariasis Loiasis RiverBlindness Canine Heartworm Infection Schistosomiasis Swimmer's ItchOriental Lung Fluke Oriental Liver Fluke Fascioliasis FasciolopsiasisOpisthorchiasis Tapeworm Infections Hydatid Disease Alveolar HydatidDisease Viral Diseases: Measles Subacute sclerosing panencephalitisCommon Cold Mumps Rubella Roseola Fifth Disease Chickenpox Respiratorysyncytial virus infection Croup Bronchiolitis Infectious MononucleosisPoliomyelitis Herpangina Hand-Foot-and-Mouth Disease Bornholm DiseaseGenital Herpes Genital Warts Aseptic Meningitis Myocarditis PericarditisGastroenteritis Acquired Immunodeficiency Syndrome (AIDS) HumanImmunodeficiency Virus (HIV) Reye's Syndrome Kawasaki Syndrome InfluenzaBronchitis Viral “Walking” Pneumonia Acute Febrile Respiratory DiseaseAcute pharyngoconjunctival fever Epidemic keratoconjunctivitis HerpesSimplex Virus 1 (HSV-1) Herpes Simplex Virus 2 (HSV-2) ShinglesCytomegalic Inclusion Disease Rabies Progressive MultifocalLeukoencephalopathy Kuru Fatal Familial Insomnia Creutzfeldt-JakobDisease Gerstmann-Straussler-Scheinker Disease Tropical SpasticParaparesis Western Equine Encephalitis California Encephalitis St.Louis Encephalitis Yellow Fever Dengue Lymphocytic choriomeningitisLassa Fever Hemorrhagic Fever Hantvirus Pulmonary Syndrome Marburg VirusInfections Ebola Virus Infections Smallpox

Multi-Drug Therapy of Infectious Diseases

Methods for treating an infectious disease are disclosed includingadministering to a patient in need thereof an Exemplary Conjugate andanother therapeutic agent that is an anti-infectious disease agent. Inone embodiment, the anti-infectious disease agent is, but not limitedto, agents listed in Table 8.

TABLE 8 β-Lactam Antibiotics: Penicillin G Penicillin V CloxacilliinDicloxacillin Methicillin Nafcillin Oxacillin Ampicillin AmoxicillinBacampicillin Azlocillin Carbenicillin Mezlocillin PiperacillinTicarcillin Aminoglycosides: Amikacin Gentamicin Kanamycin NeomycinNetilmicin Streptomycin Tobramycin Macrolides: AzithromycinClarithromycin Erythromycin Lincomycin Clindamycin Tetracyclines:Demeclocycline Doxycycline Minocycline Oxytetracycline TetracyclineQuinolones: Cinoxacin Nalidixic Acid Fluoroquinolones: CiprofloxacinEnoxacin Grepafloxacin Levofloxacin Lomefloxacin Norfloxacin OfloxacinSparfloxacin Trovafloxicin Polypeptides: Bacitracin Colistin Polymyxin BSulfonamides: Sulfisoxazole Sulfamethoxazole Sulfadiazine SulfamethizoleSulfacetamide Miscellaneous Antibacterial Agents: TrimethoprimSulfamethazole Chloramphenicol Vancomycin Metronidazole QuinupristinDalfopristin Rifampin Spectinomycin Nitrofurantoin Antiviral Agents:General Antiviral Agents: Idoxuradine Vidarabine Trifluridine AcyclovirFamcicyclovir Pencicyclovir Valacyclovir Gancicyclovir FoscarnetRibavirin Amantadine Rimantadine Cidofovir Antisense OligonucleotidesImmunoglobulins Inteferons Drugs for HIV infection: TenofovirEmtricitabine Zidovudine Didanosine Zalcitabine Stavudine LamivudineNevirapine Delavirdine Saquinavir Ritonavir Indinavir Nelfinavir

EXAMPLES Example 1 Preparation of Compound AB

Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat. No.6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M) andto this solution was added a diethylamine (60 mL). The reaction wasmonitored by HPLC and found to be complete in 2 h. The reaction mixturewas concentrated and the resulting residue was precipitated using ethylacetate (ca. 100 mL) under sonication over for 10 min. Ether (200 mL)was added and the precipitate was further sonicated for 5 min. Thesolution was allowed to stand for 30 min. without stirring and was thenfiltered and dried under high vacuum to provide Val-cit-PAB-OH, whichwas used in the next step without further purification. Yield: 8.84 g(96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) andthe resulting solution was treated with MC-OSu (Willner et al., (1993)Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction wascomplete according to HPLC after 2 h. The reaction mixture wasconcentrated and the resulting oil was precipitated using ethyl acetate(50 mL). After sonicating for 15 min, ether (400 mL) was added and themixture was sonicated further until all large particles were broken up.The solution was then filtered and the solid dried to provide anoff-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9[M−H]

Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq., U.S. Pat. No.6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M) andto this solution was added a diethylamine (60 mL). The reaction wasmonitored by HPLC and found to be complete in 2 h. The reaction mixturewas concentrated and the resulting residue was precipitated using ethylacetate (ca. 100 mL) under sonication over for 10 min. Ether (200 mL)was added and the precipitate was further sonicated for 5 min. Thesolution was allowed to stand for 30 min. without stirring and was thenfiltered and dried under high vacuum to provide Val-cit-PAB-OH, whichwas used in the next step without further purification. Yield: 8.84 g(96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) andthe resulting solution was treated with MC-OSu (Willner et al., (1993)Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction wascomplete according to HPLC after 2 h. The reaction mixture wasconcentrated and the resulting oil was precipitated using ethyl acetate(50 mL). After sonicating for 15 min, ether (400 mL) was added and themixture was sonicated further until all large particles were broken up.The solution was then filtered and the solid dried to provide anoff-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9[M−H].

The off-white solid intermediate (8.0 g, 14.0 mmol) was diluted with DMF(120 mL, 0.12 M) and to the resulting solution was addedbis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and DIEA (3.66mL, 21.0 mmol, 1.5 eq.). The reaction was complete in 1 h according toHPLC. The reaction mixture was concentrated to provide an oil that wasprecipitated with EtOAc, and then triturated with EtOAc (ca. 25 mL). Thesolute was further precipitated with ether (ca. 200 mL) and trituratedfor 15 min. The solid was filtered and dried under high vacuum toprovide Compound AB which was 93% pure according to HPLC and used in thenext step without further purification. Yield: 9.7 g (94%).

Example 2 Preparation of Compound 1

Phenylalanine t-butyl ester HCl salt (868 mg, 3 mmol), N-Boc-Dolaproine(668 mg, 1 eq.), DEPC (820 μL, 1.5 eq.), and DIEA (1.2 mL) were dilutedwith dichloromethane (3 mL). After 2 hours (h) at room temperature(about 28 degrees Celsius), the reaction mixture was diluted withdichloromethane (20 mL), washed successively with saturated aqueous(aq.) NaHCO₃ (2×10 mL), saturated aq. NaCl (2×10 mL). The organic layerwas separated and concentrated. The resulting residue was re-suspendedin ethyl acetate and was purified via flash chromatography in ethylacetate. The relevant fractions were combined and concentrated toprovide the dipeptide as a white solid: 684 mg (46%). ES-MS m/z 491.3[M+H]⁺.

For selective Boc cleavage in the presence of t-butyl ester, the abovedipeptide (500 mg, 1.28 mmol) was diluted with dioxane (2 mL). 4MHCl/dioxane (960 μL, 3 eq.) was added, and the reaction mixture wasstirred overnight at room temperature. Almost complete Boc deprotectionwas observed by RP-HPLC with minimal amount of t-butyl ester cleavage.The mixture was cooled down on an ice bath, and triethylamine (500 μL)was added. After 10 min., the mixture was removed from the cooling bath,diluted with dichloromethane (20 mL), washed successively with saturatedaq. NaHCO₃ (2×10 mL), saturated aq. NaCl (2×10 mL). The organic layerwas concentrated to give a yellow foam: 287 mg (57%). The intermediatewas used without further purification.

The tripeptide Fmoc-Meval-val-dil-O-t-Bu (prepared as described in WO02/088172, entitled “Pentapeptide Compounds and Uses Related Thereto”;0.73 mmol) was treated with TFA (3 mL), dichloromethane (3 mL) for 2 hat room temperature. The mixture was concentrated to dryness, theresidue was co-evaporated with toluene (3×20 mL), and dried in vacuumovernight. The residue was diluted with dichloromethane (5 mL) and addedto the deprotected dipeptide (287 mg, 0.73 mmol), followed by DIEA (550μL, 4 eq.), DEPC (201 μL, 1.1 eq.). After 2 h at room temperature thereaction mixture was diluted with ethyl acetate (50 mL), washedsuccessively with 10% aq. citric acid (2×20 mL), saturated aq. NaHCO₃(2×10 mL), saturated aq. NaCl (10 mL). The organic layer was separatedand concentrated. The resulting residue was re-suspended in ethylacetate and was purified via flash chromatography in ethyl acetate. Therelevant fractions were combined and concentrated to provideFmoc-Meval-val-dil-dap-phe-O-t-Bu as a white solid: 533 mg (71%). R_(f)0.4 (EtOAc). ES-MS m/z 1010.6 [M+H]⁺.

The product (200 mg, 0.2 mmol) was diluted with dichloromethane (3 mL),diethylamine (1 mL). The reaction mixture was stirred overnight at roomtemperature. Solvents were removed to provide an oil that was purifiedby flash silica gel chromatography in a step gradient 0-10% MeOH indichloromethane to provide Compound 1 as a white solid: 137 mg (87%).R_(f) 0.3 (10% MeOH/CH₂Cl₂). ES-MS m/z 788.6 [M+H]⁺.

Example 3 Preparation of Compound 2

Compound 2 was prepared from compound 1 (30 mg, 0.038 mmol) by treatmentwith 4M HCl/dioxane (4 ml) for 7 h at room temperature. The solvent wasremoved, and the residue was dried in a vacuum overnight to give provideCompound 2 as a hydroscopic white solid: 35 mg (120% calculated for HClsalt). ES-MS m/z 732.56 [M+H]⁺.

Example 4 Preparation of Compound 3

Fmoc-Meval-val-dil-dap-phe-O-t-Bu (Example 2, 50 mg) was treated with 4MHCl/dioxane (4 ml) for 16 h at room temperature. The solvent wasremoved, and the residue was dried in vacuum overnight to give 50 mg ofa hydroscopic white solid intermediate

The white solid intermediate (20 mg, 0.02 mmol) was diluted withdichloromethane (1 mL); DEPC (5 μL, 0.03 mmol, 1.5 eq.) was addedfollowed by DIEA (11 μL, 0.06 mmol, 3 eq.), and t-butylamine (3.2 μL,0.03 mmol, 1.5 eq.). After 2 h at room temperature, the reaction wasfound to be uncompleted by RP-HPLC. More DEPC (10 μL) and t-butylamine(5 μL) were added and the reaction was stirred for additional 4 h.Reaction mixture was diluted with dichloromethane (15 mL), washedsuccessively with water (5 mL), 0.1 M aq. HCl (10 mL), saturated aq.NaCl (10 mL). The organic layer was separated and concentrated. Theresulting residue was diluted with dichloromethane and purified viaflash chromatography in a step gradient 0-5% MeOH in dichloromethane.The relevant fractions were combined and concentrated to provide theFmoc protected intermediate as a white solid: 7.3 mg (36%). R_(f) 0.75(10% MeOH/CH₂Cl₂).

Fmoc protected intermediate was diluted with dichloromethane (0.5 mL)and treated with diethylamine (0.5 mL) for 3 h at room temperature. Thereaction mixture was concentrated to dryness. The product was isolatedby flash silica gel chromatography in a step gradient 0-10% MeOH indichloromethane to provide Compound 3 as a white solid: 4 mg (70%).R_(f) 0.2 (10% MeOH/CH₂Cl₂). ES-MS m/z 787 [M+H]⁺, 809 [M+Na]⁺.

Example 5 Preparation of Compound 4

Boc-L-Phenylalanine (265 mg, 1 mmol, 1 eq.) and triethyleneglycolmonomethyl ether (164 μL, 1 mmol, 1 eq.) were diluted withdichloromethane (5 mL). Then, DCC (412 mg, 2 mmol, 2 eq.) was added,followed by DMAP (10 mg). The reaction mixture was stirred overnight atroom temperature. The precipitate was filtered off. The solvent wasremoved in a vacuum, the residue was diluted with ethyl acetate, andpurified by silica gel flash chromatography in ethyl acetate. Theproduct containing fractions were pulled, concentrated, and dried invacuum to give a white solid: 377 mg (91%). R_(f) 0.5 (EtOAc). ES-MS m/z434 [M+Na]⁺.

Removal of Boc protecting group was performed by treatment of the abovematerial in dioxane (10 mL) with 4M HCl/dioxane (6 mL) for 6 h at roomtemperature. The solvent was removed in a vacuum, the residue was driedin a vacuum to give a white solid.

The HCl salt of Phenylalanine-triethyleneglycol monomethyl ether ester(236 mg, 0.458 mmol, 1 eq.) and N-Boc-Dolaproine (158 mg, 0.55 mmol, 1.2eq.) were diluted with dichloromethane (3 mL). DEPC (125 μL, 1.5 eq.)and added to the mixture followed by DIEA (250 μL, 3 eq.). After 2 h atroom temperature the reaction mixture was diluted with ethyl acetate (30mL), washed successively with saturated aq. NaHCO₃ (2×10 mL), 10% aq.citric acid (2×10 mL), saturated aq. NaCl (10 mL). The organic layer wasseparated and concentrated. The resulting residue was re-suspended inethyl acetate and was purified via flash chromatography on silica gel inethyl acetate. The relevant fractions were combined and concentrated toprovide a white foam intermediate: 131 mg (50%). R_(f) 0.25 (EtOAc).ES-MS m/z 581.3 [M+H]⁺.

Boc deprotection was done in dichloromethane (2 mL), TFA (0.5 mL) atroom temperature for 2 h. Solvent was removed in vacuum, and the residuewas co-evaporated with toluene (3×25 mL), then dried in vacuum to give138 mg of dipeptide TFA salt.

Fmoc-Meval-val-dil-OH (Example 2, 147 mg, 0.23 mmol, 1 eq.), anddipeptide TFA salt (138 mg) were diluted with dichloromethane (2 mL). Tothe mixture DEPC (63 μL, 1.5 eq.) was added, followed by DIEA (160 μL, 4eq.). After 2 h at room temperature the reaction mixture was dilutedwith dichloromethane (30 mL), washed successively with 10% aq. citricacid (2×20 mL), saturated aq. NaCl (20 mL). The organic layer wasseparated and concentrated. The resulting residue was re-suspended indichloromethane and was purified via flash chromatography on silica gelin a step gradient 0-5% MeOH in dichloromethane. The relevant fractionswere combined and concentrated to provide white foam: 205 mg (81%).R_(f) 0.4 (10% MeOH/CH₂Cl₂). ES-MS m/z 1100.6 [M+H]⁺, 1122.4 [M+Na]⁺.

Fmoc protecting group was removed by treatment with diethylamine (2 mL)in dichloromethane (6 mL). After 6 h at room temperature solvent wasremoved in vacuum, product was isolated by flash chromatography onsilica gel in a step gradient 0-10% MeOH in dichloromethane. Therelevant fractions were combined and concentrated. After evaporationfrom dichloromethane/hexane, 1:1, Compound 4 was obtained as a whitefoam: 133 mg (80%). R_(f) 0.15 (10% MeOH/CH₂Cl₂). ES-MS m/z 878.6[M+H]⁺.

Example 6 Preparation of Compound 5

Fmoc-Meval-val-dil-OH (Example 2, 0.50 g, 0.78 mmol) and dap-phe-OMe.HCl(0.3 g, 0.78 mmol, prepared according to Pettit, G. R., et al.Anti-Cancer Drug Design 1998, 13, 243-277) were dissolved in CH₂Cl₂ (10mL) followed by the addition of diisopropylethylamine (0.30 mL, 1.71mmol, 2.2 eq.). DEPC (0.20 mL, 1.17, 1.5 eq.) was added and the contentsstood over Ar. Reaction was complete according to HPLC in 1 h. Themixture was concentrated to an oil and purified by SiO₂ chromatography(300×25 mm column) and eluting with 100% EtOAc. The product was isolatedas a white foamy solid. Yield: 0.65 g (87%). ES-MS m/z 968.35 [M+H]⁺,991.34 [M+Na]⁺; UV λ_(max) 215, 265 nm.

The Fmoc-protected peptide (0.14 g, 0.14 mmol) in methylene chloride (5mL) was treated with diethylamine (2 mL) and the contents stood at roomtemperature for 2 h. The reaction, complete by HPLC, was concentrated toan oil, taken up in 2 mL of DMSO and injected into a preparative-HPLC(C₁₂-RP column, 5μ, 100 Å, linear gradient of MeCN in water (containing0.1% TFA) 10 to 100% in 40 min followed by 20 min at 100%, at a flowrate of 25 mL/min). Fractions containing the product were evaporated toafford a white powder for the trifluoroacetate salt. Yield: 0.126 g(98%). R_(f) 0.28 (100% EtOAc); ES-MS m/z 746.59 [M+H]⁺, 768.51 [M+Na]⁺;UV λ_(max) 215 nm.

Example 7 Preparation of Compound 6

The trifluoroacetate salt of Compound 5 (0.11 g, 0.13 mmol), Compound AB(0.103 g, 0.14 mmol, 1.1 eq.) and HOBt (3.4 mg, 26 μmol, 0.2 eq.) weresuspended in DMF/pyridine (2 mL/0.5 mL, respectively).Diisopropylethylamine (22.5 μL, 0.13 mmol, 1.0 eq.) was added and theyellow solution stirred while under argon. After 3 h, an additional 1.0eq. of DIEA was added. 24 hours later, 0.5 eq. of the activated linkerwas included in the reaction mixture. After 40 h total, the reaction wascomplete. The contents were evaporated, taken up in DMSO and injectedinto a prep-HPLC (C₁₂-RP column, 5μ, 100 Å, linear gradient of MeCN inwater (containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min at100%, at a flow rate of 50 mL/min). The desired fractions wereevaporated to give the product as a yellow oil. Methylene chloride (ca.2 mL) and excess ether were added to provide Compound 6 as a whiteprecipitate that was filtered and dried. Yield: 90 mg (52%). ES-MS m/z1344.32 [M+H]⁺, 1366.29 [M+Na]⁺; UV λ_(max) 215, 248 nm.

Example 8 Preparation of Compound 7

Compound 4 (133 mg, 0.15 mmol, 1 eq.), Compound AB, (123 mg, 0.167 mmol,1.1 eq.), and HOBt (4 mg, 0.2 eq.) were diluted with DMF (1.5 mL). After2 min, pyridine (5 mL) was added and the reaction was monitored usingRP-HPLC. The reaction was shown to be complete within 18 h. The reactionmixture was diluted with dichloromethane (20 mL), washed successivelywith 10% aq. citric acid (2×10 mL), water (10 mL), saturated aq. NaCl(10 mL). The organic layer was separated and concentrated. The resultingresidue was re-suspended in dichloromethane and was purified via flashchromatography on silica gel in a step gradient 0-10% MeOH indichloromethane. The relevant fractions were combined and concentratedto provide Compound 7 as a white foam: 46 mg (21%). R_(f) 0.15 (10%MeOH/CH₂Cl₂). ES-MS m/z 1476.94 [M+H]⁺.

Example 9 Preparation of MC-Val-Cit-PAB-MMAF t-butyl ester 8

Compound 1 (83 mg, 0.11 mmol), Compound AB (85 mg, 0.12 mmol, 1.1 eq.),and HOBt (2.8 mg, 21 μmol, 0.2 eq.) were taken up in dry DMF (1.5 mL)and pyridine (0.3 mL) while under argon. After 30 h, the reaction wasfound to be essentially complete by HPLC. The mixture was evaporated,taken up in a minimal amount of DMSO and purified by prep-HPLC (C₁₂-RPcolumn, 5μ, 100 Å, linear gradient of MeCN in water (containing 0.1%TFA) 10 to 100% in 40 min followed by 20 min at 100%, at a flow rate of25 mL/min) to provide Compound 8 as a white solid. Yield: 103 mg (71%).ES-MS m/z 1387.06 [M+H]⁺, 1409.04 [M+Na]⁺; UV λ_(max) 205, 248 nm.

Example 10 Preparation of MC-val-cit-PAB-MMAF 9

Compound 8 (45 mg, 32 μmol) was suspended in methylene chloride (6 mL)followed by the addition of TFA (3 mL). The resulting solution stood for2 h. The reaction mixture was concentrated in vacuo and purified byprep-HPLC (C₁₂-RP column, 5μ, 100 Å, linear gradient of MeCN in water(containing 0.1% TFA) 10 to 100% in 40 min followed by 20 min at 100%,at a flow rate of 25 mL/min). The desired fractions were concentrated toprovidemaleimidocaproyl-valine-citrulline-p-hydroxymethylaminobenzene-MMAF(MC-val-cit-PAB-MMAF) 9 as an off-white solid. Yield: 11 mg (25%). ES-MSm/z 1330.29 [M+H]⁺, 1352.24 [M+Na]⁺; UV λ_(max) 205, 248 nm.

Example 11 Preparation of MC-val-cit-PAB-MMAF tert-butyl amide 10

Compound 3 (217 mg, 0.276 mmol, 1.0 eq.), Compound AB (204 mg, 0.276mmol, 1.0 eq.), and HOBt (11 mg, 0.0828 mmol, 0.3 eq.) were diluted withpyridine/DMF (6 mL). To this mixture was added DIEA (0.048 mL), and themixture was stirred ca. 16 hr. Volatile organics were evaporated invacuo. The crude residue was purified by Chromatotron® (radialthin-layer chromatography) with a step gradient (0-5-10% methanol inDCM) to provide MC-val-cit-PAB-MMAF tert-butyl amide 10. Yield: 172 mg(45%); ES-MS m/z 1386.33 [M+H]⁺, 1408.36 [M+Na]⁺; UV λ_(max) 215, 248nm.

Example 12 Preparation of AC10-MC-MMAE by Conjugation of AC10 andMC-MMAE

AC10, dissolved in 500 mM sodium borate and 500 mM sodium chloride at pH8.0 is treated with an excess of 100 mM dithiothreitol (DTT). Afterincubation at 37° C. for about 30 minutes, the buffer is exchanged byelution over Sephadex G25 resin and eluted with PBS with 1 mM DTPA. Thethiol/Ab value is checked by determining the reduced antibodyconcentration from the absorbance at 280 nm of the solution and thethiol concentration by reaction with DTNB (Aldrich, Milwaukee, Wis.) anddetermination of the absorbance at 412 nm. The reduced antibodydissolved in PBS is chilled on ice.

The drug linker reagent, maleimidocaproyl-monomethyl auristatin E, i.e.MC-MMAE, dissolved in DMSO, is diluted in acetonitrile and water atknown concentration, and added to the chilled reduced antibody AC10 inPBS. After about one hour, an excess of maleimide is added to quench thereaction and cap any unreacted antibody thiol groups. The reactionmixture is concentrated by centrifugal ultrafiltration and AC10-MC-MMAEis purified and desalted by elution through G25 resin in PBS, filteredthrough 0.2 μm filters under sterile conditions, and frozen for storage.

Example 13 Preparation of AC10-MC-MMAF by Conjugation of AC10 andMC-MMAF

AC10-MC-MMAF was prepared by conjugation of AC 10 and MC-MMAF followingthe procedure of Example 12.

Example 14 Preparation of AC10-MC-val-cit-PAB-MMAE by Conjugation ofAC10 and MC-val-cit-PAB-MMAE

AC10-MC-val-cit-PAB-MMAE was prepared by conjugation of AC10 andMC-val-cit-PAB-MMAE following the procedure of Example 12.

Example 15 Preparation of AC10-MC- val-cit-PAB-MMAF by Conjugation ofAC10 and MC-val-cit-PAB-MMAF (9)

AC10-MC-val-cit-PAB-MMAF was prepared by conjugation of AC10 andMC-val-cit-PAB-MMAF (9) following the procedure of Example 12.

Example 16 Determination of Cytotoxicity of Selected Compounds

Cytotoxic activity of MMAF and Compounds 1-5 was evaluated on the LewisY positive cell lines OVCAR-3, H3396 breast carcinoma, L2987 lungcarcinoma and LS174t colon carcinoma Lewis Y positive cell lines can beassayed for cytotoxicity. To evaluate the cytotoxicity of Compounds 1-5,cells can be seeded at approximately 5-10,000 per well in 150 μl ofculture medium then treated with graded doses of Compounds 1-5 inquadruplicates at the initiation of assay. Cytotoxicity assays areusually carried out for 96 hours after addition of test compounds. Fiftyμl of resazurin dye may be added to each well during the last 4 to 6hours of the incubation to assess viable cells at the end of culture.Dye reduction can be determined by fluorescence spectrometry using theexcitation and emission wavelengths of 535 nm and 590 nm, respectively.For analysis, the extent of resazurin reduction by the treated cells canbe compared to that of the untreated control cells.

For 1 h exposure assays cells can be pulsed with the drug for 1 h andthen washed; the cytotoxic effect can be determined after 96 h ofincubation.

Example 17 In Vitro Cytotoxicity Cata for Selected Compounds

Table 10 shows cytotoxic effect of cAC10 Conjugates of Compounds 7-10,assayed as described in General Procedure I on a CD30+ cell line Karpas299. Data of two separate experiments are presented. The cAC10conjugates of Compounds 7 and 9 were found to be slightly more activethan cAC10-val-cit-MMAE.

TABLE 10 Conjugate IC₅₀ (ng/mL) cAC10-val-cit-MMAE 6 cAC10-7 1.0 cAC10-815 cAC10-9 0.5 cAC10-10 20

In other experiments, BR96-val-cit-MMAF was at least 250 fold morepotent than the free MMAF.

General Procedure I—Cytotoxicity Determination.

To evaluate the cytotoxicity of Exemplary Conjugates 7-10, cells wereseeded at approximately 5-10,000 per well in 150 μl of culture mediumthen treated with graded doses of Exemplary Conjugates 7-10 inquadruplicates at the initiation of assay. Cytotoxicity assays werecarried out for 96 hours after addition of test compounds. Fifty μl ofthe resazurin dye was added to each well during the last 4 to 6 hours ofthe incubation to assess viable cells at the end of culture. Dyereduction was determined by fluorescence spectrometry using theexcitation and emission wavelengths of 535 nm and 590 nm, respectively.For analysis, the extent of resazurin reduction by the treated cells wascompared to that of the untreated control cells.

Example 18 In Vitro Cell Proliferation Assay

Efficacy of ADC can be measured by a cell proliferation assay employingthe following protocol (Promega Corp. Technical Bulletin TB288; Mendozaet al. (2002) Cancer Res. 62:5485-5488):

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells(SKBR-3, BT474, MCF7 or MDA-MB-468) in medium was deposited in each wellof a 96-well, opaque-walled plate.2. Control wells were prepared containing medium and without cells.3. ADC was added to the experimental wells and incubated for 3-5 days.4. The plates were equilibrated to room temperature for approximately 30minutes.5. A volume of CellTiter-Glo Reagent equal to the volume of cell culturemedium present in each well was added.6. The contents were mixed for 2 minutes on an orbital shaker to inducecell lysis.7. The plate was incubated at room temperature for 10 minutes tostabilize the luminescence signal.8. Luminescence was recorded and reported in graphs as RLU=relativeluminescence units.

Example 19 Plasma Clearance in Rat

Plasma clearance pharmacokinetics of antibody drug conjugates and totalantibody was studied in Sprague-Dawley rats (Charles River Laboratories,250-275 gms each). Animals were dosed by bolus tail vein injection (IVPush). Approximately 300 μl whole blood was collected through jugularcannula, or by tail stick, into lithium/heparin anticoagulant vessels ateach timepoint: 0 (predose), 10, and 30 minutes; 1, 2, 4, 8, 24 and 36hours; and 2, 3, 4, 7, 14, 21, 28 days post dose. Total antibody wasmeasured by ELISA-ECD/GxhuFc-HRP. Antibody drug conjugate was measuredby ELISA-MMAE/MMAF/ECD-Bio/SA-HRP.

Example 20 Plasma Clearance in Monkey

Plasma clearance pharmacokinetics of antibody drug conjugates and totalantibody can be studied in cynomolgus monkeys. FIG. 12 shows a two-stageplasma concentration clearance study after administration ofH-MC-vc-MMAE to Cynomolgus monkeys at different doses: 0.5, 1.5, 2.5,and 3.0 mg/kg, administered at day 1 and day 21. Concentrations of totalantibody and ADC were measured over time. (H=Trastuzumab).

Example 21 Tumor Volume In Vivo Efficacy in Transgenic Explant Mice

Animals suitable for transgenic experiments can be obtained fromstandard commercial sources such as Taconic (Germantown, N.Y.). Manystrains are suitable, but FVB female mice are preferred because of theirhigher susceptibility to tumor formation. FVB males can be used formating and vasectomized CD.1 studs can be used to stimulatepseudopregnancy. Vasectomized mice can be obtained from any commercialsupplier. Founders can be bred with either FVB mice or with 129/BL6×FVBp53 heterozygous mice. The mice with heterozygosity at p53 allele can beused to potentially increase tumor formation. Some F1 tumors are ofmixed strain. Founder tumors can be FVB only.

Animals having tumors (allograft propagated from Fo5 mmtv transgenicmice) can be treated with a single or multiple dose by IV injection ofADC. Tumor volume can be assessed at various time points afterinjection.

Example 22 Synthesis of MC-MMAF Via t-Butyl Ester

MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 128.6 mg, 0.163 mmol) wassuspended in CH₂Cl₂ (0.500 mL). 6-Maleimidocaproic acid (68.9 mg, 0.326mmol) and 1,3-diisopropylcarbodiimide (0.0505 mL, 0.326 mmol) were addedfollowed by pyridine (0.500 mL). Reaction mixture was allowed to stirfor 1.0 hr. HPLC analysis indicated complete consumption of startingcompound 1. Volatile organics were evaporated under reduced pressure.Product was isolated via flash column chromatography, using a stepgradient from 0 to 5% Methanol in CH₂Cl₂. A total of 96 mg of pureMC-MeVal-Val-Dil-Dap-Phe-OtBu (12) (60% yield) was recovered. ES-MS m/z981.26 [M+H]⁺; 1003.47 [M+Na]⁺; 979.65 [M−H]⁻ See FIG. 37.

MC-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 12, 74 mg, 0.0754 mmol) wassuspended in CH₂Cl₂ (2.0 mL) and TFA (1 mL) at room temperature. After2.5 hr, HPLC analysis indicated complete consumption of startingmaterial. Volatile organics were evaporated under reduced pressure, andthe product was isolated via preparatory RP-HPLC, using a Phenomenex C₁₂Synergi Max-RP 80 Å Column (250×21.20 mm). Eluent: linear gradient 10%to 90% MeCN/0.05% TFA (aq) over 30 minutes, then isocratic 90%MeCN/0.05% TFA (aq) for an additional 20 minutes. ES-MS m/z 925.33[M+H]⁺; 947.30 [M+Na]⁺; 923.45 [M−H]⁻.

Example 23a Synthesis of MC-MMAF (11) Via Dimethoxybenzyl EsterPreparation of Fmoc-L-Phenylalanine-2,4-dimethoxybenzyl ester(Fmoc-Phe-ODMB) See FIG. 38.

A 3-neck, 5-L round-bottom flask was charged with Fmoc-L-Phenylalanine(200 g, 516 mmol Bachem), 2,4-dimethoxybenzyl alcohol (95.4 g, 567 mmol,Aldrich), and CH₂Cl₂ (2.0 L). N,N-dimethylformamide t-butyl acetal (155mL, 586 mmol, Fluka) was added to the resulting suspension over 20 minunder N₂, which resulted in a clear solution. The reaction was thenstirred at room temperature overnight, after which time TLC analysis(0.42, Heptane/EtOAc=2:1) indicated that the reaction was complete. Thereaction mixture was concentrated under reduced pressure to give a lightyellow oil, which was redissolved in CH₂Cl₂ (200 mL) and purifiedthrough a short plug of silica gel (25 cm×25 cm, CH₂Cl₂) to give acolorless foam (250 g). MeCN (1 L) was added into the resulting foam,which totally dissolved the batch. It was then concentrated to drynessand redissolved in MeCN (1 L) and the resulting suspension was stirredfor 1 h, filtered and the filter cake was rinsed with MeCN (2×200 mL) togive Fmoc-L-phenylalanine-2,4-dimethoxybenzyl ester as a white solid(113.58 g, 41%, 95.5% AUC by HPLC analysis). Data: HPLC.

Preparation L-Phenylalanine-2,4-dimethoxybenzyl ester (Phe-ODMB)

A 500-mL round-bottom flask was charged withFmoc-L-phenylalanine-2,4-dimethoxybenzyl ester (26.00 g, 48.3 mmol),CH₂Cl₂ (150 mL) and diethylamine (75 mL, Acros). Mixture was stirred atroom temperature and the completion monitored by HPLC. After 4 h, themixture was concentrated (bath temp <30° C.). The residue wasresuspended in CH₂Cl₂ (200 mL) and concentrated. This was repeated once.To the residue was added MeOH (20 mL), which caused the formation of agel. This residue was diluted with CH₂Cl₂ (200 mL), concentrated and thecloudy oil left under vacuum overnight. The residue was suspended inCH₂Cl₂ (100 mL), then toluene (120 mL) was added. The mixture wasconcentrated and the residue left under vacuum overnight.

Data: HPLC, ¹H NMR.

Preparation of Fmoc-Dolaproine (Fmoc-Dap)

Boc-Dolaproine (58.8 g, 0.205 mol) was suspended in 4 N HCl in1,4-dioxane (256 mL, 1.02 mol, Aldrich). After stirring for 1.5 hours,TLC analysis indicated the reaction was complete (10% MeOH/CH₂Cl₂) andthe mixture was concentrated to near-dryness. Additional 1,4-dioxane wascharged (50 mL) and the mixture was concentrated to dryness and driedunder vacuum overnight. The resulting white solid was dissolved in H₂O(400 mL) and transferred to a 3-L, three-neck, round-bottom flask with amechanical stirrer and temperature probe. N,N-diisopropylethylamine(214.3 mL, 1.23 mol, Acros) was added over one minute, causing anexotherm from 20.5 to 28.2° C. (internal). The mixture was cooled in anice bath and 1,4-dioxane was added (400 mL). A solution of Fmoc-OSu(89.90 g, 0.267 mol, Advanced ChemTech) in 1,4-dioxane (400 mL) wasadded from an addition funnel over 15 minutes, maintaining the reactiontemperature below 9° C. The mixture was allowed to warm to roomtemperature and stir for 19 hours, after which the mixture wasconcentrated by rotary evaporation to an aqueous slurry (390 g). Thesuspension was diluted with H₂O (750 mL) and Et₂O (750 mL), causing acopious white precipitate to form. The layers were separated, keepingthe solids with the organic layer. The aqueous layer was acidified usingcone. HCl (30 mL) and extracted with EtOAc (3×500 mL). The combinedextracts were dried over MgSO₄, filtered and concentrated to give 59.25g of a yellow oil A. The Et₂O extract was extracted once with sat.NaHCO₃ (200 mL), keeping the solids with the aqueous layer. The aqueoussuspension was acidified using conc. HCl (50 mL) and extracted with Et₂O(50 mL) keeping the solids with the organic layer. The organic layer wasfiltered and concentrated to give 32.33 g of a yellow oil B. The twooils (A and B) were combined and purified by flash chromatography onsilica gel eluting with CH₂Cl₂ (3.5 L), then 3% MeOH/CH₂Cl₂ (9 L) togive 68.23 g of Fmoc-dolaproine as a white foam (81%, 97.5% purity byHPLC (AUC)).

Preparation of Fmoc-Dap-Phe-ODMB

Crude Phe-ODMB (48.3 mmol) was suspended in anhydrous DMF (105 mL,Acros) for 5 minutes and Fmoc-Dap (19.80 g, 48.3 mmol) was added. Themixture was cooled in an ice bath and TBTU (17.08 g, 53.20 mmol, MatrixInnovations) was added. N,N-diisopropylethylamine (25.3 mL, 145.0 mmol,Acros) was added via syringe over 3 min. After 1 h, the ice bath wasremoved and the mixture was allowed to warm over 30 min. The mixture waspoured into water (1 L) and extracted with ethyl acetate (300 mL). Afterseparation, the aqueous layer was re-extracted with ethyl acetate (2×150mL). The combined organic layers were washed with brine (150 mL), dried(MgSO4) and filtered (filter paper) to remove the insolubles (inorganicsand some dibenzofulvene). After concentration, the residue (41 g) wasadsorbed on silica (41 g) and purified by chromatography (22 cm×8 cmcolumn; 65% Heptane/EtOAc (2.5 L); 33% Heptane/EtOAc (3.8 L), to give29.4 g of product as a white foam (86%, 92% purity by HPLC).

Data: HPLC, ¹H NMR, TLC (1:1 EtOAc/Heptane Rf=0.33, red in vanillinstain).

Preparation of Dap-Phe-ODMB

A 1-L round bottom flask was charged with Fmoc-Dap-Phe-ODMB (27.66 g),CH₂Cl₂ (122 mL) and diethylamine (61 mL, Acros). The solution wasstirred at room temperature and the completion monitored by HPLC. After7 h, the mixture was concentrated (bath temp. <30° C.). The residue wassuspended in CH₂Cl₂ (300 mL) and concentrated. This was repeated twice.To the residue was added MeOH (20 mL) and CH₂Cl₂ (300 mL), and thesolution was concentrated. The residue was suspended in CH₂Cl₂ (100 mL)and toluene (400 mL), concentrated, and the residue left under vacuumovernight to give a cream-like residue.

Data: HPLC, ¹H NMR, MS.

Preparation of Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB

Crude Dap-Phe-ODMB (39.1 mmol) was suspended in anhydrous DMF (135 mL,Acros) for 5 minutes and Fmoc-MeVal-Val-Dil-OH (24.94 g, 39.1 mmol, seeExample 2 for preparation) was added. The mixture was cooled in an icebath and TBTU (13.81 g, 43.0 mmol, Matrix Innovations) was added.N,N-Diisopropylethylamine (20.5 mL, 117.3 mmol, Acros) was added viasyringe over 2 minutes. After 1 hour, the ice bath was removed and themixture was allowed to warm over 30 min. The mixture was poured intowater (1.5 L) and diluted with ethyl acetate (480 mL). After standingfor 15 minutes, the layers were separated and the aqueous layer wasextracted with ethyl acetate (300 mL). The combined organic layers werewashed with brine (200 mL), dried (MgSO₄) and filtered (filter paper) toremove insolubles (inorganics and some dibenzofulvene). Afterconcentration, the residue (49 g) was scraped from the flask andadsorbed on silica (49 g) and purified by chromatography (15 cm×10 cmdia column; 2:1 EtOAc/Heptane (3 L), EtOAc (5 L); 250 mL fractions) togive 31.84 g of Fmoc-MeVal-Val-Dil-Dap-Phe-ODMB as a white foam (73%,93% purity by HPLC (AUC)).

Data: HPLC, TLC (2:1 EtOAc/heptane, Rf=0.21, red in vanillin stain).

Preparation of MeVal-Val-Dil-Dap-Phe-ODMB

A 1-L, round-bottom flask was charged withFmoc-MeVal-Val-Dil-Dap-Phe-ODMB (28.50 g), CH₂Cl₂ (80 mL) anddiethylamine (40 mL). Mixture was stirred at room temperature overnightand then was concentrated under reduced pressure. The residue wasadsorbed on silica (30 g) and purifed by flash chromatography (15 cm×8cm dia column; 2% MeOH/DCM (2 L), 3% MeOH/DCM (1 L), 6% MeOH/DCM (4 L);250 mL fractions) to give 15.88 g of MeVal-Val-Dil-Dap-Phe-ODMB as awhite foam (69%, 96% purity by HPLC (AUC)).

Data: HPLC, TLC (6% MeOH/DCM, Rf=0.24, red in vanillin stain).

Preparation of MC-MeVal-Val-Dil-Dap-Phe-ODMB

A 50-mL, round-bottom flask was charged with MeVal-Val-Dil-Dap-Phe-ODMB(750 mg, 0.85 mmol), anhydrous DMF (4 mL), maleimidocaproic acid (180mg, 0.85 mmol), and TBTU (300 mg, 0.93 mmol, Matrix Innovations) at roomtemperature. N,N-Diisopropylethylamine (450 μL, 2.57 mmol) was added viasyringe. After 1.5 hours, the mixture was poured in water (50 mL) anddiluted with ethyl acetate (30 mL). NaCl was added to improve theseparation. After separation of the layers, the aqueous layer wasextracted with ethyl acetate (25 mL). The combined organic layers weredried (MgSO4), filtered and concentrated. The resulting oil (1 g) waspurified by flash chromatography [100 mL silica; 25% Heptane/EtOAc (100mL), 10% Heptane/EtOAc (200 mL), EtOAc (1.5 L)] to giveMC-MeVal-Val-Dil-Dap-Phe-ODMB (13) as a white foam (521 mg, 57%, 94%purity by HPLC(AUC)).

Data: 1H NMR, HPLC.

Preparation of MC-MeVal-Val-Dil-Dap-Phe-OH (MC-MMAF)(11)

A 50-mL, round-bottom flask was charged withMC-MeVal-Val-Dil-Dap-Phe-ODMB (Compound 13, 428 mg, 0.39 mmol) anddissolved in 2.5% TFA/CH₂Cl₂ (20 mL). The solution turned pink-purpleover 2 min. The completion was monitored by HPLC and TLC (6% MeOH/DCM,KMnO₄ stain). After 40 min, three drops of water were added and thecloudy pink-purple mixture was concentrated to give 521 mg of a pinkresidue. Purification by chromatography (15% IPA/DCM) gave 270 mg ofMC-MMAF (73%, 92% purity by HPLC) as a white solid.

Example 23b Synthesis of Analog of Mc-MMAF

MeVal-Val-Dil-Dap-Phe-OtBu (compound 1, 35 mg, 0.044 mmol) was suspendedin DMF (0.250 mL). 4-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-benzoic acid(11 mg, 0.049 mmol) and HATU (17 mg, 0.044 mmol) were added followed byDIEA (0.031 mL, 0.17 mmol) See FIG. 39. This reaction mixture wasallowed to stir for 2.0 hr. HPLC analysis indicated complete consumptionof starting compound 1.

Product was isolated via preparatory RP-HPLC, using a Phenomenex C₁₂Synergi Max-RP 80 Å Column (250×21.20 mm). Eluent: linear gradient 10%to 80% MeCN/0.05% TFA (aq) over 8 minutes, then isocratic 80% MeCN/0.05%TFA (aq) for an additional 12 minutes. A total of 20 mg of pure product(14) was isolated (0.02 mmol, 46% yield). ES-MS m/z 987.85 [M+H]⁺;1019.41 [M+Na]⁺; 985.54 [M−H]⁻.

MB-MeVal-Val-Dil-Dap-Phe-OtBu (Compound 14, 38 mg, 0.0385 mmol) wassuspended in CH₂Cl₂ (1 mL) and TFA (1 mL). Mixture was stirred for 2.0hr, and then volatile organics were evaporated under reduced pressure.Product was purified by preparatory RP-HPLC, using a Phenomenex C₁₂Synergi Max-RP 80 Å Column (250×21.20 mm). Eluent: linear gradient 10%to 80% MeCN/0.05% TFA (aq) over 8 minutes, then isocratic 80% MeCN/0.05%TFA (aq) for an additional 12 minutes. A total of 14.4 mg of MB-MMAFproduct was isolated (0.015 mmol, 40% yield). ES-MS m/z 930.96 [M+H]⁺952.98 [M+Na]⁺; 929.37 [M−H]⁻.

Example 23c Preparation of MC-MeVal-Cit-PAB-MMAF (16)

To a room temperature suspension of Fmoc-MeVal-OH (3.03 g, 8.57 mmol)and N,N′-disuccimidyl carbonate (3.29 g, 12.86 mmol) in CH₂Cl₂ (80 mL)was added DIEA (4.48 mL, 25.71 mmol). This reaction mixture was allowedto stir for 3.0 hr, and then poured into a separation funnel where theorganic mixture was extracted with 0.1 M HCl (aq). The crude organicresidue was concentrated under reduced pressure, and the product wasisolated by flash column chromatography on silica gel using a 20-100%ethyl acetate/hexanes linear gradient. A total of 2.18 g of pureFmoc-MeVal-OSu (4.80 mmoles, 56% yield) was recovered.

To a room temperature suspension of Fmoc-MeVal-OSu (2.18 g, 4.84 mmol)in DME (13 mL) and THF (6.5 mL) was added a solution of L-citrulline(0.85 g, 4.84 mmol) and NaHCO₃ (0.41 g, 4.84 mmol) in H₂O (13 mL). Thesuspension was allowed to stir at room temperature for 16 hr, then itwas extracted into tert-BuOH/CHCl₃/H₂O, acidified to pH=2-3 with 1 MHCl. The organic phase was separated, dried and concentrated underreduced pressure. The residue was triturated with diethyl etherresulting in 2.01 g of Fmoc-MeVal-Cit-COOH which was used withoutfurther purification.

The crude Fmoc-MeVal-Cit-COOH was suspended in 2:1 CH₂Cl₂/MeOH (100 mL),and to it was added p-aminobenzyl alcohol (0.97 g, 7.9 mmol) and EEDQ(1.95 g, 7.9 mmol). This suspension was allowed to stir for 125 hr, thenthe volatile organics were removed under reduced pressure, and theresidue was purified by flash column chromatography on silica gel usinga 10% MeOH/CH₂Cl₂. Pure Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896 mmol, 18.5%yield) was recovered. ES-MS m/z 616.48 [M+H]⁺.

To a suspension of Fmoc-MeVal-Cit-PAB-OH (0.55 g, 0.896 mmol) in CH₂Cl₂(40 mL) was added STRATOSPHERES™ (piperizine-resin-bound) (>5 mmol/g,150 mg). After being stirred at room temperature for 16 hr the mixturewas filtered through celite (pre-washed with MeOH), and concentratedunder reduced pressure. Residue was triturated with diethyl ether andhexanes. Resulting solid material, MeVal-Cit-PAB-OH, was suspended inCH₂Cl₂ (20 mL), and to it was added MC-OSu (0.28 g, 0.896 mmol), DIEA(0.17 mL, 0.99 mmol), and DMF (15 mL). This suspension was stirred for16 hr, but HPLC analysis of the reaction mixture indicated incompletereaction, so the suspension was concentrated under reduced pressure to avolume of 6 mL, then a 10% NaHCO₃ (aq) solution was added and thesuspension stirred for an additional 16 hr. Solvent was removed underreduced pressure, and the residue was purified by flash columnchromatography on silica gel using a 0-10% MeOH/CH₂Cl₂ gradient,resulting in 42 mg (0.072 mmol, 8% yield) of MC-MeVal-Cit-PAB-OH.

To a suspension of MC-MeVal-Cit-PAB-OH (2.37 g, 4.04 mmol) andbis(nitrophenyl)carbonate (2.59 g, 8.52 mmol) in CH₂Cl₂ (10 mL) wasadded DIEA (1.06 mL, 6.06 mmol). This suspension was stirred for 5.5 hr,concentrated under reduced pressure and purified by trituration withdiethyl ether. MC-MeVal-Cit-PAB-OCO-pNP (147 mg, 0.196 mmol) wassuspended in a 1:5 pyridine/DMF solution (3 mL), and to it was addedHOBt (5 mg, 0.039 mmol), DIEA (0.17 mL, 0.978 mmol) and MMAF (compound2, 150 mg, 0.205 mmol). This reaction mixture was stirred for 16 hr atroom temperature, and then purified by preparatory RP-HPLC (×3), using aPhenomenex C₁₂ Synergi Max-RP 80 Å Column (250×21.20 mm). Eluent: lineargradient 10% to 90% MeCN/0.05% TFA (aq) over 30 minutes, then isocratic90% MeCN/0.05% TFA (aq) for an additional 20 minutes.MC-MeVal-Cit-PAB-MMAF (16) was obtained as a yellowish solid (24.5 mg,0.0182, 0.45% yield). ES-MS m/z 1344.95 [M+H]⁺; 1366.94 [M+Na]⁺.

Example 23c Preparation of succinimide ester of suberyl-Val-Cit-PAB-MMAF(17)

Compound 1 (300 mg, 0.38 mmol), Fmoc-Val-Cit-PAB-pNP (436 mg, 0.57 mmol,1.5 eq.) were suspended in anhydrous pyridine, 5 mL. HOBt (10 mg, 0.076mmol, 0.2 eq.) was added followed by DIEA (199 μl, 1.14 mmol, 3 eq.).Reaction mixture was sonicated for 10 min, and then stirred overnight atroom temperature. Pyridine was removed under reduced pressure, residuewas re-suspended in CH₂Cl₂. Mixture was separated by silica gel flashchromatography in a step gradient of MeOH, from 0 to 10%, in CH₂Cl₂.Product containing fractions were pulled, concentrated, dried in vacuumovernight to give 317 mg (59% yield) of Fmoc-Val-Cit-PAB-MMAF-OtBu.ES-MS m/z 1415.8 [M+H]⁺.

Fmoc-Val-Cit-PAB-MMAF-OtBu (100 mg) was stirred in 20% TFA/CH₂Cl₂ (10mL), for 2 hrs. Mixture was diluted with CH₂Cl₂ (50 mL). Organic layerwas washed successively with water (2×30 mL) and brine (1×30 mL).Organic phase was concentrated, loaded onto pad of silica gel in 10%MeOH/CH₂Cl₂. Product was eluted with 30% MeOH/CH₂Cl₂. After drying invacuum overnight, Fmoc-Val-Cit-PAB-MMAF was obtained as a white solid,38 mg, 40% yield. ES-MS m/z 1357.7 [M−H]⁻.

Fmoc-Val-Cit-PAB-MMAF, 67 mg, was suspended in CH₂Cl₂ (2 mL)diethylamine (2 mL) and DMF (2 mL). Mixture was stirred for 2 hrs atroom temperature. Solvent was removed under reduced pressure. Residuewas co-evaporated with pyridine (2 mL), then with toluene (2×5 mL),dried in vacuum. Val-Cit-PAB-MMAF was obtained as brownish oil, and usedwithout further purification.

All Val-Cit-PAB-MMAF prepared from 67 mg of Fmoc-Val-Cit-PAB-MMAF, wassuspended in pyridine (2 mL), and added to a solution of disuccinimidylsuberate (74 mg, 0.2 mmol, 4 eq.), in pyridine (1 mL). Reaction mixturewas stirred at room temperature. After 3 hrs ether (20 mL) was added.Precipitate was collected, washed with additional amount of ether.Reddish solid was suspended in 30% MeOH/CH₂Cl₂, filtered trough a pad ofsilica gel with 30% MeOH/CH₂Cl₂ as an eluent. Compound 17 was obtainedas white solid, 20 mg (29% yield). ES-MS m/z 1388.5 [M−H]⁻

Example 24 In Vivo Efficacy of mcMMAF Antibody-Drug Conjugates

Efficacy of cAC10-mcMMAF in Karpas-299 ALCL Xenografts:

To evaluate the in vivo efficacy of cAC10-mcMMAF with an average of 4drug moieties per antibody (cAC10-mcF4), Karpas-299 human ALCL cellswere implanted subcutaneously into immunodeficient C.B-17 SCID mice(5×10⁶ cells per mouse). Tumor volumes were calculated using the formula(0.5×L×W²) where L and W are the longer and shorter of two bidirectionalmeasurements. When the average tumor volume in the study animals reachedapproximately 100 mm³ (range 48-162) the mice were divided into 3 groups(5 mice per group) and were either left untreated or were given a singleintravenous injection through the tail vein of either 1 or 2 mg/kgcAC10-mcF4 (FIG. 1). The tumors in the untreated mice grew rapidly to anaverage volume of >1,000 mm³ within 7 days of the start of therapy. Incontrast, all of the cAC10-mcF4 treated tumor showed rapid regressionwith 3/5 in the 1 mg/kg group and 5/5 in the 2 mg/kg group obtainingcomplete tumor response. While the tumor in one of the completeresponders in the 2 mg/kg group did recur approximately 4 weeks later,there were no detectable tumors in the remaining 4/5 responders in thisgroup and in the 3 complete responders in the 1 mg/kg group at 10 weekspost therapy.

Efficacy of cBR96-mcMMAF in L2987 NSCLC Xenografts:

cBR96 is a chimeric antibody that recognizes the Le^(Y) antigen. Toevaluate the in vivo efficacy of cBR96-mcMMAF with 4 drugs per antibody(cBR96-mcF4) L2987 non-small cell lung cancer (NSCLC) tumor fragmentswere implanted into athymic nude mice. When the tumors averagedapproximately 100 mm³ the mice were divided into 3 groups: untreated and2 therapy groups. For therapy, as shown in FIG. 3 a, mice wereadministered cBR96-mcF4 at either 3 or 10 mg/kg/injection every 4 daysfor a total of 4 injections (q4dx4). As shown in FIG. 3 b, mice wereadministered cBR96-mcF4 or a non-binding control conjugate, cAC10-mcF4,at 10 mg/kg/injection every 4 days for a total of 4 injections (q4dx4).As shown in FIGS. 3 a and 3 b, BR96-mcF4 produced pronounced tumorgrowth delay compared to the controls.

FIG. 2 shows an in vivo, single dose, efficacy assay of cAC10-mcMMAF insubcutaneous L540CY. For this study there were 4 mice in the untreatedgroup and 10 in each of the treatment groups.

Example 25 In Vitro Efficacy of MC-MMAF Antibody-Drug Conjugates

Activity of cAC10-Antibody-Drug Conjugates Against CD30⁺ Cell Lines.

FIGS. 4 a and 16 b show dose-response curves from a representativeexperiment where cultures of Karpas 299 (anaplastic large cell lymphoma)and L428 (Hodgkin's Lymphoma) were incubated with serially dilutedcAC10-mcMMAF (FIG. 4 a) or cAC10-vcMMAF (FIG. 4 b) for 96 hours. Thecultures were labeled for 4 hours with 50 μM resazurin[7-hydroxy-3H-phenoxazin-3-one 10-oxide] and the fluorescence measured.The data were reduced in GraphPad Prism version 4.00 using the4-parameter dose-response curve fit procedure. IC₅₀ values are definedas the concentration where growth is reduced 50% compared with untreatedcontrol cultures. Each concentration was tested in quadruplicate.

Activity of cBR96-Antibody-Drug Conjugates Against Le^(y+) Cell Lines.

FIGS. 5 a and 5 b show dose-response curves from a representativeexperiment where cultures of H3396 (breast carcinoma) and L2987 (nonsmall cell lung carcinoma) were incubated with serially dilutedcBR96-mcMMAF (FIG. 5 a) or −vcMMAF (FIG. 5 b) for 96 hours. The cultureswere labeled for 4 hours with 50 μM resazurin and the fluorescencemeasured. The data were reduced in GraphPad Prism version 4.00 using the4-parameter dose-response curve fit procedure. IC₅₀ values are definedas the concentration where growth is reduced 50% compared with untreatedcontrol cultures. Each concentration is tested in quadruplicate.

Activity of c1F6-Antibody-Drug Conjugates Against CD70⁺ Renal CellCarcinoma Cell Lines.

FIGS. 6 a and 6 b show dose-response curves from a representativeexperiment where cultures of Caki-1 and 786-O cells were incubated withserially diluted c1F6-mcMMAF (FIG. 6 a) or −vcMMAF (FIG. 6 b) for 96hours. The cultures were labeled for 4 hours with 50 μM resazurin andthe fluorescence measured. The data were reduced in GraphPad Prismversion 4.00 using the 4-parameter dose-response curve fit procedure.IC₅₀ values are defined as the concentration where growth is reduced 50%compared with untreated control cultures. Each concentration is testedin quadruplicate.

Example 26 Purification of Trastuzumab

One vial containing 440 mg HERCEPTIN® (huMAb4D5-8, rhuMAb HER2, U.S.Pat. No. 5,821,337) antibody) was dissolved in 50 mL MES buffer (25 mMMES, 50 mM NaCl, pH 5.6) and loaded on a cation exchange column(Sepharose S, 15 cm×1.7 cm) that had been equilibrated in the samebuffer. The column was then washed with the same buffer (5 columnvolumes). Trastuzumab was eluted by raising the NaCl concentration ofthe buffer to 200 mM. Fractions containing the antibody were pooled,diluted to 10 mg/mL, and dialyzed into a buffer containing 50 mmpotassium phosphate, 50 mM NaCl, 2 mM EDTA, pH 6.5.

Example 27 Preparation of Trastuzumab-MC-MMAE by Conjugation ofTrastuzumab and MC-MMAE

Trastuzumab, dissolved in 500 mM sodium borate and 500 mM sodiumchloride at pH 8.0 is treated with an excess of 100 mM dithiothreitol(DTT). After incubation at 37° C. for about 30 minutes, the buffer isexchanged by elution over Sephadex G25 resin and eluted with PBS with 1mM DTPA. The thiol/Ab value is checked by determining the reducedantibody concentration from the absorbance at 280 nm of the solution andthe thiol concentration by reaction with DTNB (Aldrich, Milwaukee, Wis.)and determination of the absorbance at 412 nm. The reduced antibodydissolved in PBS is chilled on ice.

The drug linker reagent, maleimidocaproyl-monomethyl auristatin E(MMAE), i.e. MC-MMAE, dissolved in DMSO, is diluted in acetonitrile andwater at known concentration, and added to the chilled reduced antibodytrastuzumab in PBS. After about one hour, an excess of maleimide isadded to quench the reaction and cap any unreacted antibody thiolgroups. The reaction mixture is concentrated by centrifugalultrafiltration and trastuzumab-MC-MMAE is purified and desalted byelution through G25 resin in PBS, filtered through 0.2 μm filters understerile conditions, and frozen for storage.

Example 28 Preparation of Trastuzumab-MC-MMAF by Conjugation ofTrastuzumab and MC-MMAF

Trastuzumab-MC-MMAF was prepared by conjugation of trastuzumab andMC-MMAF following the procedure of Example 27.

Example 29 Preparation of Trastuzumab-MC- Val-Cit-PAB-MMAE byConjugation of Trastuzumab and MC-val-cit-PAB-MMAE

Trastuzumab-MC-val-cit-PAB-MMAE was prepared by conjugation oftrastuzumab and MC-val-cit-PAB-MMAE following the procedure of Example27.

Example 30 Preparation of Trastuzumab-MC- Val-Cit-PAB-MMAF byConjugation of Trastuzumab and MC-Val-Cit-PAB-MMAF 9

Trastuzumab-MC-val-cit-PAB-MMAF was prepared by conjugation oftrastuzumab and MC-val-cit-PAB-MMAF 9 following the procedure of Example27.

Example 31 Rat Toxicity

The acute toxicity profile of free drugs and ADC was evaluated inadolescent Sprague-Dawley rats (75-125 gms each, Charles RiverLaboratories (Hollister, CA). Animals were injected on day 1, completechemistry and hematology profiles were obtained at baseline, day 3 andday 5 and a complete necropsy was performed on day 5. Liver enzymemeasurements was done on all animals and routine histology as performedon three random animals for each group for the following tissues:sternum, liver, kidney, thymus, spleen, large and small intestine. Theexperimental groups were as follows:

μg MMAF/ MMAF/ Group Administered mg/kg m² MAb N/Sex 1 Vehicle 0 0 0 2/F2 trastuzumab-MC-val-cit- 9.94 840 4.2 6/F MMAF 3trastuzumab-MC-val-cit- 24.90 2105 4.2 6/F MMAF 4 trastuzumab-MC(Me)-10.69 840 3.9 6/F val-cit-PAB-MMAF 5 trastuzumab-MC(Me)- 26.78 2105 3.96/F val-cit-PAB-MMAF 6 trastuzumab-MC-MMAF 10.17 840 4.1 6/F 7trastuzumab-MC-MMAF 25.50 2105 4.1 6/F 8 trastuzumab-MC-val-cit- 21.852105 4.8 6/F PAB-MMAF For trastuzumab-MC-val-cit-MMAF,trastuzumab-MC(Me)-val-cit-PAB-MMAF, trastuzumab-MC-MMAF andtrastuzumab-MC-val-cit-PAB-MMAF, the μg MMAF/m² was calculated using731.5 as the MW of MMAF and 145167 as the MW of Herceptin.

The body surface area was calculated as follows: [{(body weight in gramsto 0.667 power)×11.8}/10000]. (Guidance for Industry and Reviewers,2002).

The dose solutions were administered by a single intravenous bolustail-vein injection on Study Day 1 at a dose volume of 10 mL/kg. Bodyweights of the animals were measured pre-dose on Study Day 1 and dailythereafter. Whole blood was collected into EDTA containing tubes forhematology analysis. Whole blood was collected into serum separatortubes for clinical chemistry analysis. Blood samples were collectedpre-dose on Study Day −4, Study Day 3 and Study Day 5. Whole blood wasalso collected into sodium heparin containing tubes at necropsy and theplasma was frozen at −70° C. for possible later analysis. The followingtissues were collected and placed in neutral buffered formalin atnecropsy: liver, kidneys, heart, thymus, spleen, brain, sternum andsections of the GI tract, including stomach, large and small intestine.Sternum, small intestine, large intestine, liver, thymus, spleen andkidney were examined.

Liver associated serum enzyme levels at each timepoint were compared toa range (5th and 95th percentile) from normal female Sprague-Dawleyrats. White blood cell and platelet counts at each timepoint werecompared to a range (5th and 95th percentile) from normal femaleSprague-Dawley rats.

High dose study in normal female Sprague-Dawley rats:

Group 1: Vehicle Group 2: trastuzumab-MC-MMAF, 52.24 mg/kg, 4210 μg/m²Group 3: trastuzumab-MC-MMAF, 68.25 mg/kg, 5500 μg/m² Group 4:trastuzumab-MC-MMAF, 86.00 mg/kg, 6930 μg/m²

Tissues from 11 animals were submitted for routine histology. Theseanimals had been part of an acute dose-ranging toxicity study using atrastuzumab-MC-MMAF immunoconjugate. Animals were followed for 12 daysfollowing dosing.

Example 32 Cynomolgus Monkey Toxicity/Safety

Three groups of four (2 male, 2 female) naive Macaca fascicularis(cynomolgus monkey) were studied for trastuzumab-MC-vc-PAB-MMAE andtrastuzumab-MC-vc-PAB-MMAF. Intravenous administration was conducted atdays 1 and 22 of the studies.

Sample Group Dose Vehicle 1 day 1 1M/1F day 22 H-MC-vc-PAB-MMAE 2  180μg/m² (0.5 mg/kg) at day 1 2M/2F 1100 μg/m² (3.0 mg/kg) at day 22H-MC-vc-PAB-MMAE 3  550 μg/m² (1.5 mg/kg) at day 8 2M/2F  550 μg/m² (1.5mg/kg) at day 29 H-MC-vc-PAB-MMAE 4  880 μg/m² (2.5 mg/kg) at day 152M/2F  880 μg/m² (2.5 mg/kg) at day 36 Vehicle 1 day 1 1M/1F day 22H-MC-vc-PAB-MMAF 2  180 μg/m² (0.5 mg/kg) at day 1 2M/2F 1100 μg/m² (3.0mg/kg) at day 22 H-MC-vc-PAB-MMAF 3  550 μg/m² (1.5 mg/kg) at day 12M/2F  550 μg/m² (1.5 mg/kg) at day 22 H-MC-vc-PAB-MMAF 4  880 μg/m²(2.5 mg/kg) at day 1 2M/2F  880 μg/m² (2.5 mg/kg) at day 22 H =trastuzumab

Dosing is expressed in surface area of an animal so as to be relevant toother species, i.e. dosage at μg/m² is independent of species and thuscomparable between species. Formulations of ADC contained PBS, 5.4 mMsodium phosphate, 4.2 mM potassium phosphate, 140 mM sodium chloride, pH6.5.

Blood was collected for hematology analysis predose, and at 5 min., 6hr, 10 hr, and 1, 3, 5, 7, 14, 21 days after each dose. Erythrocyte(RBC) and platelet (PLT) counts were measured by the light scatteringmethod. Leukocyte (WBC) count was measured by the peroxidase/basophilmethod. Reticulocyte count was measured by the light scattering methodwith cationic dye. Cell counts were measured on an Advia 120 apparatus.ALT (alanine aminotransferase) and AST (aspartate aminotransferase) weremeasured in U/L by UV/NADH; IFCC methodology on an Olympus AU400apparatus, and using Total Ab ELISA-ECD/GxhuFc-HRP. Conj. AbELISA-MMAE/MMAF//ECD-Bio/SA-HRP tests.

Example 33 Production, Characterization and Humanization of Anti-ErbB2Monoclonal Antibody 4D5

The murine monoclonal antibody 4D5 which specifically binds theextracellular domain of ErbB2 was produced as described in Fendly et al.(1990) Cancer Research 50:1550-1558. Briefly, NIH 3T3/HER2-3₄₀₀ cells(expressing approximately 1×10⁵ ErbB2 molecules/cell) produced asdescribed in Hudziak et al. Proc. Natl. Acad. Sci. (USA) 84:7158-7163(1987) were harvested with phosphate buffered saline (PBS) containing 25mM EDTA and used to immunize BALB/c mice. The mice were given injectionsi.p. of 10⁷ cellsin 0.5 ml PBS on weeks 0, 2, 5 and 7. The mice withantisera that immunoprecipitated ³²P-labeled ErbB2 were given i.p.injections of a wheat germ agglutinin-Sepharose (WGA) purified ErbB2membrane extract on weeks 9 and 13. This was followed by an i.v.injection of 0.1 ml of the ErbB2 preparation and the splenocytes werefused with mouse myeloma line X63-Ag8.653. Hybridoma supernatants werescreened for ErbB2-binding by ELISA and radioimmunoprecipitation.

Epitope Mapping and Characterization

The ErbB2 epitope bound by monoclonal antibody 4D5 was determined bycompetitive binding analysis (Fendly et al. Cancer Research 50:1550-1558(1990)). Cross-blocking studies were done by direct fluorescence onintact cells using the PANDEXTM Screen Machine to quantitatefluorescence. The monoclonal antibody was conjugated with fluoresceinisothiocyanate (FITC), using established procedures (Wofsy et al.Selected Methods in Cellular Immunology, p. 287, Mishel and Schiigi(eds.) San Francisco: W.J. Freeman Co. (1980)). Confluent monolayers ofNIH 3T3/HER2-3₄₀₀ cells were trypsinized, washed once, and resuspendedat 1.75×10⁶ cell/ml in cold PBS containing 0.5% bovine serum albumin(BSA) and 0.1% NaN₃. A final concentration of 1% latex particles (IDC,Portland, Oreg.) was added to reduce clogging of the PANDEXTM platemembranes. Cells in suspension, 20 μl, and 20 μl of purified monoclonalantibodies (100 μm/ml to 0.1 μg/ml) were added to the PANDEX™ platewells and incubated on ice for 30 minutes. A predetermined dilution ofthe FITC-labeled monoclonal antibody in 20 μl was added to each well,incubated for 30 minutes, washed, and the fluorescence was quantitatedby the PANDEX™ Monoclonal antibodies were considered to share an epitopeif each blocked binding of the other by 50% or greater in comparison toan irrelevant monoclonal antibody control. In this experiment,monoclonal antibody 4D5 was assigned epitope I (amino acid residues fromabout 529 to about 625, inclusive within the ErbB2 extracellular domain.

The growth inhibitory characteristics of monoclonal antibody 4D5 wereevaluated using the breast tumor cell line, SK-BR-3 (see Hudziak et al.(1989) Molec. Cell. Biol. 9(3):1165-1172). Briefly, SK-BR-3 cells weredetached by using 0.25% (vol/vol) trypsin and suspended in completemedium at a density of 4×10⁵ cells per ml. Aliquots of 100 μl (4×10⁴cells) were plated into 96-well microdilution plates, the cells wereallowed to adhere, and 100 μl of media alone or media containingmonoclonal antibody (final concentration 5 μg/ml) was then added. After72 hours, plates were washed twice with PBS (pH 7.5), stained withcrystal violet (0.5% in methanol), and analyzed for relative cellproliferation as described in Sugarman et al. (1985) Science230:943-945. Monoclonal antibody 4D5 inhibited SK-BR-3 relative cellproliferation by about 56%.

Monoclonal antibody 4D5 was also evaluated for its ability to inhibitHRG-stimulated tyrosine phosphorylation of proteins in the M_(r) 180,000range from whole-cell lysates of MCF7 cells (Lewis et al. (1996) CancerResearch 56:1457-1465). MCF₇ cells are reported to express all knownErbB receptors, but at relatively low levels. Since ErbB2, ErbB3, andErbB4 have nearly identical molecular sizes, it is not possible todiscern which protein is becoming tyrosine phosphorylated whenwhole-cell lysates are evaluated by Western blot analysis. However,these cells are ideal for HRG tyrosine phosphorylation assays becauseunder the assay conditions used, in the absence of exogenously addedHRG, they exhibit low to undetectable levels of tyrosine phosphorylationproteins in the M_(r) 180,000 range.

MCF7 cells were plated in 24-well plates and monoclonal antibodies toErbB2 were added to each well and incubated for 30 minutes at roomtemperature; then rHRGβ1₁₇₇₋₂₄₄ was added to each well to a finalconcentration of 0.2 nM, and the incubation was continued for 8 minutes.Media was carefully aspirated from each well, and reactions were stoppedby the addition of 100 μl of SDS sample buffer (5% SDS, 25 mM DTT, and25 mM Tris-HCl, pH 6.8). Each sample (25 μl) was electrophoresed on a4-12% gradient gel (Novex) and then electrophoretically transferred topolyvinylidene difluoride membrane. Antiphosphotyrosine (4G10, from UBI,used at 1 μg/ml) immunoblots were developed, and the intensity of thepredominant reactive band at M_(r□)1180,000 was quantified byreflectance densitometry, as described previously (Holmes et al. (1992)Science 256:1205-1210; Sliwkowski et al. J. Biol. Chem. 269:14661-14665(1994)).

Monoclonal antibody 4D5 significantly inhibited the generation of aHRG-induced tyrosine phosphorylation signal at M_(r) 180,000. In theabsence of HRG, but was unable to stimulate tyrosine phosphorylation ofproteins in the M_(r) 180,000 range. Also, this antibody does notcross-react with EGFR (Fondly et al. Cancer Research 50:1550-1558(1990)), ErbB3, or ErbB4. Monoclonal antibody 4D5 was able to block HRGstimulation of tyrosine phosphorylation by □50%.

The growth inhibitory effect of monoclonal antibody 4D5 on MDA-MB-175and SK-BR-3 cells in the presence or absence of exogenous rHRGβ1 wasassessed (Schaefer et al. Oncogene 15:1385-1394 (1997)). ErbB2 levels inMDA-MB-175 cells are 4-6 times higher than the level found in normalbreast epithelial cells and the ErbB2-ErbB4 receptor is constitutivelytyrosine phosphorylated in MDA-MB-175 cells. Monoclonal antibody 4D5 wasable to inhibit cell proliferation of MDA-MB-175 cells, both in thepresence and absence of exogenous HRG. Inhibition of cell proliferationby 4D5 is dependent on the ErbB2 expression level (Lewis et al. CancerImmunol. Immunother. 37:255-263 (1993)). A maximum inhibition of 66% inSK-BR-3 cells could be detected. However this effect could be overcomeby exogenous HRG.

The murine monoclonal antibody 4D5 was humanized, using a “geneconversion mutagenesis” strategy, as described in U.S. Pat. No.5,821,337, the entire disclosure of which is hereby expresslyincorporated by reference. The humanized monoclonal antibody 4D5 used inthe following experiments is designated huMAb4D5-8. This antibody is ofIgG1 isotype.

REFERENCES CITED

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodiments thatare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart and are intended to fall within the scope of the appended claims.

All references cited herein are incorporated by reference in theirentirety and for all purposes to the same extent as if each individualpublication or patent or patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

What is claimed is:
 1. A method for treating cancer comprisingadministering to a patient in need thereof an effective amount of anantibody-drug conjugate having Formula Ia′:AbA_(a)-W_(w)—Y_(y)-D)_(p)  Ia′ or a pharmaceutically acceptable saltthereof, wherein: Ab is an antibody that specifically binds to anantigen expressed by the cancer, A is a Stretcher unit, a is 1, each Wis independently an Amino Acid unit, w is an integer ranging from 2 to12, Y is a Spacer unit, and y is 1 or 2, p ranges from 1 to about 20,and D is a drug moiety of Formula D_(F):

wherein the wavy line of D_(F) indicates the covalent attachment site toY: R² is selected from H and C₁-C₈ alkyl; R³ is selected from H, C₁-C₈alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈ alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈carbocycle), C₃-C₈ heterocycle and C₁-C₈ alkyl-(C₃-C₈ heterocycle); R⁴is selected from H, C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈alkyl-aryl, C₁-C₈ alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈alkyl-(C₃-C₈ heterocycle); R⁵ is selected from H and methyl; or R⁴ andR⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(n)— wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl and C₃-C₈ carbocycle and n is selected from 2, 3, 4,5 and 6; R⁶ is selected from H and C₁-C₈ alkyl; R⁷ is selected from H,C₁-C₈ alkyl, C₃-C₈ carbocycle, aryl, C₁-C₈ alkyl-aryl, C₁-C₈alkyl-(C₃-C₈ carbocycle), C₃-C₈ heterocycle and C₁-C₈ alkyl-(C₃-C₈heterocycle); each R⁸ is independently selected from H, OH, C₁-C₈ alkyl,C₃-C₈ carbocycle and O—(C₁-C₈ alkyl); R⁹ is selected from H and C₁-C₈alkyl; R¹⁰ is selected from aryl or C₃-C₈ heterocycle; Z is O, S, NH, orNR¹², wherein R¹² is C₁-C₈ alkyl; R¹¹ is selected from H, C₁-C₂₀ alkyl,aryl, C₃-C₈ heterocycle, —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂; m isan integer ranging from 1-1000; R¹³ is C₂-C₈ alkyl; R¹⁴ is H or C₁-C₈alkyl; each occurrence of R¹⁵ is independently H, COOH,—(CH₂)_(n)—N(R¹⁶)₂, —(CH₂)_(n)—SO₃H, or —(CH₂)_(n)—SO₃—C₁-C₈ alkyl; eachoccurrence of R¹⁶ is independently H, C₁-C₈ alkyl, or —(CH₂), —COOH; andn is an integer ranging from 0 to
 6. 2. The method of claim 1, whereinthe antibody-drug conjugate has the formula:

or a pharmaceutically acceptable salt thereof, wherein R¹⁷ is C₁-C₁₀alkylene-, —C₃-C₈ carbocyclo-, —O—(C₁-C₈ alkyl)-, -arylene-, —C₁-C₁₀alkylene-arylene-, -arylene-C₁-C₁₀ alkylene-, —C₁-C₁₀ alkylene-(C₃-C₈carbocyclo)-, —(C₃-C₈ carbocyclo)-C₁-C₁₀alkylene-, —C₃-C₈ heterocyclo-,—C₁-C₁₀ alkylene-(C₃-C₈ heterocyclo)-, —(C₃-C₈ heterocyclo)-C₁-C₁₀alkylene-, —(CH₂CH₂O)_(r)—, and —(CH₂CH₂O)_(r)—CH₂—; and r is an integerranging from 1-10.
 3. The method of claim 1, wherein the antibody-drugconjugate has the formula:

or a pharmaceutically acceptable salt thereof.
 4. The method of claim 3,wherein the antibody-drug conjugate has the formula:

or a pharmaceutically acceptable salt thereof.
 5. The method of claim 1,wherein D has the formula:

or a pharmaceutically acceptable salt thereof.
 6. The method of claim 1,wherein Z is —O— and R¹¹ is —H, methyl or t-butyl
 7. The method of claim6, wherein R¹¹ is —H.
 8. The method of claim 1, wherein R² and R⁶ areeach methyl, and R⁹ is —H.
 9. The method of claim 1, wherein theantibody-drug conjugate has the formula:

or a pharmaceutically acceptable salt thereof; wherein Val is valine,and Cit is citrulline.
 10. The method of claim 9, wherein the antibodyis a monoclonal antibody.